bluuebunny/crossref_metadata_2025 huggingface dataset:
Vascular O2.‐ and H2O2 production and oxidative stress resistance in two closely related rodent species with disparate longevity
Authors: Csiszar Anna , Labinskyy Nazar , Ballabh Praveen , Austad Steven N , Podlutsky Andrej , Ungvari Zoltan
Published: June 2021

Vascular aging is characterized by increased oxidative stress, impaired NO bioavailability and enhanced apoptotic cell death. The oxidative stress hypothesis of aging predicts that vascular cells of long‐lived species exhibit lower production of ROS and/or superior resistance to oxidative stress. We tested this hypothesis using two taxonomically related rodents, the white‐footed mouse (Peromyscus leucopus) and M. musculus (lifespan: 8 vs. 3.5 years). Compared to mice, in aortas of P. leucopus there were 1) a lower NADPH oxidase expression/activity, endothelial O2.‐/H2O2 production and ROS generation by mitochondria, 2) increased expression of catalase, Gpx‐1, HO‐1 and eNOS, and 3) increased NO production. In mouse aortas treatment with ox‐LDL elicited oxidative stress, endothelial dysfunction and apoptosis, whereas vessels of P. leucopus were resistant to deleterious effects of oxLDL and H2O2. Primary fibroblasts from P. leucopus also exhibited less H2O2 ‐induced DNA damage. Thus, increased lifespan potential in P. leucopus is associated with a decreased cellular ROS generation and increased oxidative stress resistance, which accord with the prediction of the oxidative stress hypothesis of aging.
Resveratrol attenuates cigarette smoking‐induced pro‐inflammatory alterations in the endothelial phenotype
Authors: Ungvari Zoltan , Labinskyy Nazar , Pacher Pal , Zhang Cuihua , Podlutsky Andrej , Csiszar Anna
Published: June 2021

Previously we have shown that water soluble components of cigarette smoke (CS) up‐regulate NAD(P)H oxidases and activate NF‐kB leading to vascular inflammation, likely increasing increased cardiovascular risk in smokers. To determine whether the sirtuin activator resveratrol (RES) protects endothelial cells against the deleterious effects of CS, rats were exposed to the smoke of 5 cigarettes per day (for one week, with or without RES treatment). CS impaired acetylcholine‐induced relaxations of carotid arteries, which could be improved by RES treatment. Both smoking and in vitro treatment of coronary arterial endothelial cells (CAECs) with CS extract significantly increased endothelial O2.‐ production, which were attenuated by RES. Expression of the pro‐inflammatory cytokines (IL‐1, IL‐6 and TNFa) and that of iNOS was significantly increased by CS, which could be attenuated by RES treatment. In CAECs CS treatment elicited NF‐kB activation and increased monocyte adhesiveness, which were prevented by RES. Thus, RES at nutritionally relevant concentrations attenuates CS‐induced oxidative stress, improves endothelial function and inhibits vascular inflammation.
Adjuvant and novel anti‐cancer drug, keyhole limpet hemocyanin, targets serotonin signaling pathway by inhibiting feeding, locomotion and egg laying in C. elegans
Authors: Huggins Luke Graham , Davis Linda Vona
Published: June 2021

Keyhole limpet hemocyanin (KLH) is a copper‐containing respiratory pigment found in the mollusk, Megathura crenulata. Its primary biological role is the uptake, transport, and release of oxygen during respiration. KLH stimulates a host immune response making it suitable as an adjuvant for vaccines, and has been shown to inhibit cellular proliferation in human cancer cell lines of the breast, esophagus, pancreas, and prostate. Little is known about the physiological effects and molecular targets of this novel immunotherapy agent. To study the functional characterization of KLH, we selected Caenorhabditis elegans, a small, free‐living nematode worm frequently used as a genetic and developmental model organism. Worms were treated for 0–48 hours in liquid culture containing KLH (0–500 mg/mL), and scored for changes in feeding, locomotion, and egg‐laying behaviors. When treated with the drug KLH, there was a time and dose‐dependent decrease in feeding behavior and locomotion coupled with an increase in egg‐retention. All behaviors are regulated by serotonin signaling in C. elegans through a heterotrimeric G‐protein linked receptor pathway. These results suggest that the molecular actions of KLH target the serotonergic signaling pathway and warrant further validation as a novel therapeutic entry point in a disease model. Funded by NIH AI034544 and P20 RR16477.
Pyruvate‐fortified cardioplegia evokes novel myocardial expression of erythropoietin in swine undergoing cardiopulmonary bypass
Authors: Ryou Myoung Gwi , Flaherty Devin C , Hoxha Besim , Sun Jie , Ferlitch Heather R , Olivencia‐Yurvati Albert H , Mallet Robert T
Published: June 2021

Background: Fortification of cardioplegia solution with pyruvate, an energy substrate and potent antioxidant, markedly increased post‐surgical cardiac contractile function and shortened post‐surgical hospitalization in patients recovering from coronary artery revascularization on cardiopulmonary bypass (CPB). Pyruvate's favorable effects persist after its clearance from the circulation. Pyruvate is reported to block degradation of the α subunit of the transcription factor hypoxia‐inducible factor 1 (HIF‐1).

Hypothesis: Pyruvate‐enhanced cardioplegia provokes HIF‐1‐driven expression of the cytoprotective cytokine erythropoietin (EPO).

Methods: Following sternotomy and cannulation, hearts were arrested in situ for 60 min with 4:1 blood:crystalloid cardioplegia, while pigs were maintained on CPB. The crystalloid component contained 24 mM lactate or pyruvate. After 30 min cardiac reperfusion with cardioplegia‐free blood, the pigs were weaned from CPB. Left ventricular myocardium was sampled at 4 h recovery for immunoblot measurements of HIF‐1α, EPO and actin loading control, and reverse transcriptase PCR detection of EPO mRNA.

Results: Pyruvate‐fortified cardioplegia dramatically increased myocardial EPO mRNA abundance vs. conventional, lactate cardioplegia. Pyruvate increased EPO and HIF‐1α protein contents by 62 and 32%, respectively (P< 0.05).

Conclusion: For the first time, EPO expression was detected in myocardium. Pyruvate‐fortified cardioplegia markedly enhanced this novel and potentially cardioprotective EPO expression, at least in part by stabilizing HIF‐1. Support: Osteopathic Heritage Foundation grant 02‐18‐522
Normalization of the myocardial metabolic phenotype precedes the cardiac contractile recovery and morphological reverse remodeling during post‐heart failure recompensation in chronic dogs
Authors: Qanud Khaled R , Gravel John , Mamdani Mohammed , Kozicky Mark , Shimran George , Lee Daniel , Gupte Sachin A , Recchia Fabio A
Published: June 2021

We tested the hypothesis that full functional recovery requires the normalization of the myocardial metabolic phenotype in post‐heart failure recompensation. In 6 chronically instrumented dogs, heart failure (HF) was induced by pacing the left ventricle (LV) at high frequency for 4 weeks. When LV end‐diastolic pressure reached 23±1 mmHg, the pacemaker was turned off to allow 10 days for recompensation. The comparisons were made with 5 dogs in HF and 5 control dogs. Cardiac free fatty acids and glucose oxidation were measured by infusing 3H‐oleate and 14C‐glucose intravenously. After recompensation, hemodynamic parameters returned to control values, however dP/dtmax and LV ejection fraction remained, respectively, 15.8±5.3 and 14.6±2.4% lower than control (both P<0.05) and the heart weight to bodyweight ratio was increased (9.9±0.5 vs 8.0±0.1 g/kg, P<0.05). Despite this incomplete functional and morphological recovery, fatty acid and glucose oxidation returned to control values and therefore they were, compared to HF, respectively higher (45.1±7.1 vs 20.4±7.7 nmol/min/g, P<0.05) and lower (28.9±7.8 vs 61.6±15.9 nmol/min/g, P<0.05). No significant differences in cardiac oxygen consumption were found among groups. In conclusion, in post‐HF recompensation, the normalization of cardiac substrate utilization is dissociated from complete functional recovery and reverse morphological remodeling. Supported by P01‐HL‐74237.
Regulation of Gene Expression During Hypoxia Using the HL‐1 Cardiomyocyte Cell Line
Authors: Jackson Kelly , Paris James , Virtanen Carl , Takahashi Mark
Published: June 2021

Currently, heart disease is the leading cause of death in the United States. Often, this involves ischemic injury to heart tissue arising from coronary occlusions. As this is a very complex disease, a need has arisen for a representative model to study the adaptive mechanisms that lead to changes in this tissue. We have utilized a unique cardiac cell line (HL‐1) as a model of adult cardiomyocytes and have carried out microarray analysis of these cells as a means to prove their use as a model. Messenger RNA expression was first characterized and of the 15, 264 clones profiled, 614 were statistically found to be differentially expressed. From this list, 163 have no gene associated with them, 141 were ESTs and 75 were identified as RIKEN clones. The remaining 235 clones belong to a variety of genes with very diverse functions ranging from metabolic to cell cycle regulation. Hypoxia‐inducible factor (HIF‐1) is a well documented transcription factor that responds quickly to conditions of low oxygen. We have used chromatin immunoprecipitation on microarrays to further examine the role of HIF‐1 during both transient and chronic hypoxia at the transcriptional level. Preliminary results have demonstrated distinct temporal cohorts of genes regulated by HIF‐1 that can be linked to changes observed at the mRNA level. The results of this study will provide initial insights into the mechanisms leading to changes at the cellular level as a result of low oxygen and may provide leads into understanding the changes that occur during heart disease. This work was supported by the ORF.
Calcium‐dependent protein kinase C alpha and the frequency‐dependent increase in phosphorylation of troponin I in failing hearts
Authors: Lamberts Regis R. , Hamdani Nazha , Walker Lori A. , de Tombe Pieter P. , van der Velden Jolanda , Stienen Ger J. M.
Published: June 2021

In healthy myocardium the force‐frequency relation, one of the key factors modulating cardiac performance, is positive, while in failing myocardium a negative relation is found. This difference has been ascribed to changes in calcium homeostasis and, more recently, to changes in calcium sensitivity through increased phosphorylation of troponin I (TnI). Our objective was to determine whether altered activation of calcium‐dependent protein kinase C alpha (PKCα) determines the increase in phosphorylation of TnI with frequency in failing hearts.

Right ventricular heart failure was induced by monocrotaline (MCT; 80mg/kg). After 4 weeks isovolumic pressure–frequency relations were determined in Langendorff‐perfused hearts in a control (n=19) and MCT‐treated group (n=19). Thereafter, hearts were kept quiescent or paced at 9 Hz for 60 min and flash frozen to determine phosphorylation of TnI and PKCα by 1D‐gel electrophoresis.

The pressure–frequency relation was constant in control and negative in MCT group. Phosphorylation of TnI did not depend on frequency in control but increased in MCT group. Protein expression and phosphorylation levels of PKCα were both increased with frequency in MCT group.

These results indicate that frequency‐dependent changes in PKC‐phosphorylatable sites of TnI in failing hearts originate from an altered balance in calcium‐dependent kinase/phosphatase activity.
Role of T‐type Ca2+ channel in hypernatremic precondition
Authors: Pastukh Viktor , Ricci Craig , Wu Songwei , Gillespie Mark N. , Schaffer Stephen W.
Published: June 2021

Hypernatremia is a well established risk factor for heart disease. However, the actions of salt excess are complex and their interactions with ischemic injury remain unclear. The present study examined whether chronic extracellular sodium treatment could precondition the cardiomyocyte against hypoxia‐mediated cell death. Cardiomyocytes were incubated during the pre‐hypoxic period with medium containing either 167 mM Na+(hypernatremia), 50 mM Mannitol (hyperosmotic) or 142 mM Na+(control). Hypernatremia rendered the cells resistant to hypoxia‐mediated cell death, an effect linked to a significant reduction in Ca2+ accumulation. The reduction in [Ca2+]I was associated with decreased T‐type Ca2+ current but not L‐type Ca2+ current. This effect appeared to be specific for hypernatremia, as there was no change in the T‐type Ca2+ channel in the hyperosmotically treated cells. Downregulation of the Cav3.1 variant of the T‐type Ca2+ channel using iRNA also led to a reduction in hypoxia‐mediated Ca2+ accumulation and cell death. Therefore, downregulation of the Cav3.1 variant is cytoprotective and contributes to the development of hypernatremic preconditioning.
Different LV and RV response to α‐adrenergic stimulation in vivo
Authors: Huang Janice , Lu Li , Ye Shuyu , Xu Ya , Ahmad Hasan , Schwartz Gregory G. , Baker Anthony J. , Greyson Clifford R.
Published: June 2021

Objective: α‐adrenergic agonists (α‐Adr) exert positive cardiac inotropic effects. Recent data suggest that rat left ventricle (LV) and right ventricle (RV) respond differently to α‐Adr in vitro. The purpose of this study is to determine whether α‐Adr exerts different effects on the LV and RV in vivo.

Methods: Pigs (n=8) were instrumented with micromanometers and sonomicrometry crystals in the LV and RV. Contractility was determined by preload‐adjusted regional external work, a loading condition‐independent index, and regional blood flow by microspheres. Measurements were obtained at baseline, after CaCl2 15 mg‐kg−1 bolus, and during phenylephrine (PHE) 3–6 μg‐kg−1‐min−1 IV for 60 min.

Results: LV and RV contractility both increased similarly after CaCl2 (LV: 143±7% of baseline; RV: 145±10% of baseline); in contrast, PHE increased LV but decreased RV contractility (LV: 147±10% of baseline; RV: 90±19% of baseline; p<0.05 for paired difference) despite increased LV and RV pressure. LV and RV regional blood flow both increased during PHE (LV: 0.8±0.2 and RV 0.6±0.1 ml‐gm−1‐min−1 at baseline; LV: 1.5±0.2 and RV: 1.0±0.2 ml‐gm−1‐min−1 during PHE).

Conclusions: The α‐Adr agent phenylephrine exerts positive inotropic effects on the LV but negative inotropic effects on the RV in vivo. This difference is not due to differences in loading conditions or regional myocardial blood flow. Supported by NHLBI.
Effect of simulated microgravity on mesenteric blood flow during orthostatic challenge
Authors: Kaufman Susan Elaine , Levasseur Jody
Published: June 2021

Astronauts, particularly females, suffer from post‐flight orthostatic hypotension. We proposed that microgravity impairs reflex control of the splanchnic circulation during an orthostatic challenge. Female rats were trained to mount a 45% incline (Head up tilt, HUT). They were then implanted with transit time flow probes on the superior mesenteric artery (SMA) and vein (SMV). After 7 days recovery, mesenteric arterial and venous blood flow was measured during HUT. After hindlimb unloading (HU) for 14 days, the responses to HUT were then retested. Baseline SMA and SMV blood flows were 13.7±3.5 and 9.7±0.1mL/min respectively. During HUT these fell (At 5 sec: Δ SMA flow = −7mL/min, Δ SMV flow = −3mL/min; At 10 sec: Δ SMA flow = −4mL/min, Δ SMV flow = −3mL/min). There was thus an immediate mesenteric arterial constriction, and a net loss of ~4mL/min blood from the mesenteric vascular bed. After 14 days HU, there were no such changes in blood flow during HUT i.e. there was no reflex mesenteric arterial constriction and no mobilization of venous blood. We propose microgravity impairs splanchnic control of cardiac preload and afterload during HUT, and contributes to orthostatic hypotension. Canadian Space Agency.
Hindlimb unweighting induces changes in the rat abdominal aorta RhoA‐Rho kinase pathway
Authors: Summers Scott M. , Nguyen Steven V. , Purdy Ralph E.
Published: June 2021

Hindlimb unweighting (HU) treatment in rats mimics the cephalic fluid shift experienced by astronauts in microgravity. It may also serve as a model for ground based orthostatic hypotension. It has been shown previously that the abdominal aorta of the HU rat exhibits a deficit in contractile capacity in response to adrenergic agonists. Wistar rats were subjected to 20 days of HU treatment and their abdominal aortas were removed and sectioned into 3 mm rings. These rings and those from paired with age matched controls were subjected to concentration‐contractile response curves to either U46619 or phenylephrine with and without the Rho kinase inhibitor, Y27632. Alternatively, rings were snap frozen after exposure to 1 μM U46619 or 1 μM phenylephrine and Western blot analysis was performed. HU treatment decreased contractile response to U46619, a known activator of the Rho Pathway and decreased the inhibitory effect of Y27632. Western blotting revealed decreased levels of RhoA after HU treatment as well as decreased phosphorylation of both the 110 Kd myosin light chain phosphatase regulatory subunit after phenylephrine stimulation and myosin light chain after both U46619 and phenylephrine stimulation. It is concluded that the RhoA‐Rho kinase pathway is altered in the abdominal aorta of the HU rat and this may contribute to the HU‐induced decrease in contraction. Funded by NASA Grant NN04CK29G.
WISE‐2005: Lower Body Negative Pressure Treadmill and Resistive Exercise Countermeasures Maintain Physiologic Function in Women during 60‐days of Simulated Microgravity
Authors: Macias Brandon Richard , Schneider Suzanne , Lee Stuart MC , Guinet Patrick , Hughson Richard , Smith Scott , Watenpaugh Donald , Hargens Alan
Published: June 2021

INTRODUCTION: We hypothesized that supine LBNP treadmill exercise combined with Flywheel resistive exercise maintains upright physiologic responses following 60‐days of head‐down tilt (HDT) bed rest (BR). METHODS: 16 healthy women (age 25–40 years) underwent 60‐days HDT (‐6°) BR. Women were assigned to either a non‐exercise control group (CON, n=8) or to an exercise group (EX, n=8). EX subjects performed a 40‐min, variable intensity LBNP exercise protocol at foot‐ward forces between 1.0–1.1 times body weight, followed by 10‐min of resting LBNP 3–4 days/week. Resistive exercise of maximal concentric and eccentric supine leg press and heel raise exercises were performed using a flywheel ergometer 2–3 days/week. IRBs approved this study with informed/written consent. RESULTS: Post‐BR VO2pk was not different in EX (−3.3±1.2%) but decreased significantly in CON (−21.2±2.1%), p< 0.05. Post‐BR orthostatic tolerance time (mean±se) decreased significantly less in EX (19.3±1.3 to 14.4±1.5 min) than in CON (17.5±0.1 to 9.1±1.5 min), p=0.03. Post‐BR muscle strength decreased significantly in CON, but was preserved in EX. Post‐BR bone resorption was greater than pre‐BR in both groups (p<0.05). Bone formation markers, were significantly elevated (p<0.05) in EX than in CON. CONCLUSIONS: Supine LBNP treadmill exercise along with flywheel resistive exercise maintains upright exercise capacity, orthostatic responses and muscle strength during 60‐days HDT BR. Supported by ESA, CSA, CNES; and NASA: NNJ04HF71G and NNJ04HF72G.
Strain distribution within human triceps surae muscles during isometric contraction: in vivo study by Tagging MRI
Authors: Oda Toshiaki , Kinugasa Ryuta , Rajendra Mayoran , Himeno Ryutaro , Kataoka Hiroyuki , Yokota Hideo , Yamamoto Akio , Kanehisa Hiroaki , Fukunaga Tetsuo , Kawakami Yasuo
Published: June 2021

Sizable strain induces injuries of biological tissues. Non‐uniform distribution of strain in a contracting muscle has been predicted with computer simulation, with no actual data available for living human muscles. The study objective was to determine in vivo local strain of human triceps surae muscles during isometric contraction with tagging MRI. Quasi‐sagittal MR images with grid‐tagging of soft tissues were obtained while the subject (n=6 men) repeated sub‐maximal plantar flexions at 40% of maximal voluntary contraction force. Local deformation of muscle tissues was determined from the displacement of each cross grid traced by a pattern‐matching program. The local strain was computed from the displacements based on finite element theory. In all subjects, considerable intramuscular non‐uniform strain was observed in both the soleus and gastrocmenius muscles. Strain over 20% concentrated on the origin of the soleus muscle, and large strains were also observed near the boundaries of each muscle and between muscle and bone. Individual differences in strain were considerably large both in magnitude and distribution. Strain concentration on a specific part could be related with the site‐specificity of the occurrence of muscle injury.
The effect of resistance training on systemic inflammatory markers in middle‐aged and older adults
Authors: Barnes Jill Nicole , Cortez‐Cooper Miriam Y. , Anton Maria M. , Tanaka Hirofumi
Published: June 2021

Systemic inflammation has been implicated in the pathophysiology of cardiovascular disease. There is accumulating evidence that aerobic exercise has a beneficial effect on inflammatory markers. However, it is unclear whether the same is true for resistance exercise. Resistance training is increasingly prescribed for middle‐aged and older adults to improve overall health. Currently, it is not known whether the recommended amount of resistance training is sufficient to reduce systemic inflammation in middle‐aged and older adults. Plasma samples of 27 sedentary but healthy middle‐aged and older adults (aged 54±8 yr) who had undergone 13 weeks of supervised resistance training (10 exercises at ∼70% of 1RM, 3 d/wk) or supervised stretching (attention and time control) were analyzed in this retrospective analysis. The resistance training group increased lean body mass (p<0.01) and maximum strength of the major muscle groups (p<0.05). After 13‐weeks, blood pressure and fasting glucose, cholesterol, and triglycerides did not change. Concentrations of C‐reactive protein, C‐type natriuretic peptide, and tumor necrosis factor‐α were unaltered by either intervention. Our results indicate that the amount of resistance training currently recommended is not sufficient to reduce systemic inflammatory markers in healthy middle‐aged and older adults.

Supported by NIH awards AG20966, HL072729
EXERCISE AND CLASS IIa HISTONE DEACETYLASES IN HUMAN SKELETAL MUSCLE
Authors: Hargreaves Mark , Fairlie Erin , McGee Sean , Garnham Andrew
Published: June 2021

The class IIa histone deacetylases (HDACs) are involved in the regulation of skeletal muscle phenotype via effects on myocyte enhancer factor 2 (MEF2) transcriptional activity. We have previously shown that a single exercise bout reduces nuclear HDAC5 abundance and MEF2‐HDAC5 association, with a concomitant increase in GLUT4 mRNA, in human skeletal muscle (McGee & Hargreaves. Diabetes. 53:1208–1214, 2004). In the present study, we obtained muscle (v. lateralis) samples from nine untrained men (23 ± 1 yr, 75 ± 6 kg) before and after exercise (60 min, 77 ± 2% peak pulmonary oxygen uptake) to examine exercise effects on other class IIa HDACs. There was a 40% reduction (P<0.05) in nuclear HDAC4 after exercise, while nuclear HDAC5 was reduced (P<0.05) by 13%, with no changes in total HDAC4 or HDAC5 expression. There were no changes in nuclear HDAC7 or HDAC9 abundance with exercise, nor was there a change in acetyl‐H3 (K9/K14) with exercise (1.16 ± 0.15 vs. 1.0 ± 0.0 arb. units). These results suggest that nuclear‐cytosolic shuttling of HDAC4 and HDAC5, but not HDAC7 and HDAC9, could play a role in the exercise‐induced changes in skeletal muscle gene expression.
Mechanical overload increases muscle mass but does not change myosin isoform expression in a rodent hibernator, Spermophilus lateralis
Authors: Rourke Bryan , Selpides Pocholo‐Jose , Nowell Megan , Rippen Marie , Caiozzo Vincent J , Baker Michael
Published: June 2021

Hibernating ground squirrels do not show slow to fast myosin heavy‐chain (MyHC) isoform transitions with atrophy, and we test whether increased muscle loading would also fail to produce typical MyHC isoform shifts. Field‐caught, Summer‐active ground squirrels (Spermophilus lateralis) were assessed for the responsiveness of hindlimb plantaris muscles to mechanical overload through synergist surgical ablation. Plantaris muscles of surgical animals were 40% larger than controls after 14 days, but did not show any of the expected shifts in MyHC isoform expression. We also measured MyHC isoform mRNA expression, citrate synthase activity, and the expression of a suite of genes involved in the control of skeletal muscle mass and MyHC isoform expression, including MAFbx, MuRF1, FOXO1, IGF1 and IGFBP5, and myostatin. Our results show an unusual insensitivity of hibernator muscles to mechanical overload induced isoform shifts, yet muscle mass was increased, which has implications for isoform expression and muscle adaptations during hibernation. Funding sources: NIH SCORE, NIH RISE, CSULB.
Chronic intermittent hypoxia (CIH) alters the neuronal response to norepinephrine (NE) in the pre‐Bötzinger complex (pre‐BötC)
Authors: Garcia Alfredo J. , Prabhakar Nanduri , Ramirez Jan‐Marino
Published: June 2021

The impact of CIH on mechanisms underlying the rhythmogenesis of breathing is poorly understood. This ongoing study, tests the hypothesis that CIH disrupts normal neuronal activity in the pre‐BötC. Neonatal mice (P0–P1) were exposed to ~8hrs. of intermittent hypoxia for 7–10 days. After exposure to CIH, brainstem slices containing the pre‐BötC were prepared for either electrophysiological or biochemical experiments. Extracellular recordings from the pre‐BötC show that CIH causes an increase in the irregularity of population burst frequency. Moreover, application of norepinephrine (NE) produces unstable augmentation of population burst amplitude. Intracellular recordings from within the CIH‐conditioned pre‐BötC demonstrate that individual neurons reliably firing action potentials in phase with the rhythm, intermittently fail to produce action potentials when exposed NE. These findings indicate that CIH promotes a state where the coupling of individual neurons to the rest the neuronal network is decreased leading to the unstable amplitude modulation of population bursts during exposure to NE. Biochemical analysis reveals that CIH activates HIF‐1 alpha and increases lipid peroxidation within the pre‐BötC. Hence, the changes in the neuronal response to NE may be the result of adaptations to repeated hypoxic and oxidative stresses produced by CIH.
Computational modeling indicates anaerobic glycogenolytic ATP synthesis contributes little to quadriceps energy balance during exhaustive bicycling exercise
Authors: Jeneson Jeroen , Schmitz Joep , van Riel Natal , Groenendaal Willemijn , Nicolay Klaas , Hilbers Peter
Published: June 2021

It has been proposed that anaerobic glycogenolysis may contribute as much as 20% to cellular ATP synthesis during contractile work on basis of indirect evidence from 31P NMR spectroscopic measurements of proton balance in human limb muscles (1). Here, this hypothesis was tested more rigorously using a systems biology approach. We constructed a computational model of energy metabolism in muscle composed of slow‐twitch oxidative, fast‐twitch oxidative and fast‐twitch glycolytic fibers incorporating mitochondrial and glycogenolytic ATP synthetic pathways operating in parallel. Next, we measured the in vivo dynamics of phosphocreatine (PCr), Pi, hexose monophosphates (HMP) and H+ concentrations in quadriceps muscle of human subjects performing in‐magnet bicycling exercise using gated 31P NMR spectroscopy (2). The measured PCr dynamics were then compared to model simulations for different magnitudes of glycogenolytic ATP synthetic flux. In contrast to previous indirect analyses (1), we found that anaerobic ATP synthesis contributions of only 10% or less sufficed to explain energy balance during exercise; this in spite of massive activation of glycogenolysis as evidenced by large accumulations of HMP of up to 10 mM directly following exercise (2).
Fibronectin or chitosan addition to electrospun cellulose acetate scaffolds enhances endothelial cell infiltration
Authors: Rubenstein David Alan , Zamfir Dan , Frame Mary D.
Published: June 2021

Our goal was to evaluate endothelial cell infiltration into electrospun cellulose acetate scaffolds, with a fiber diameter range of 1–5μm. Chitosan was added prior to electrospinning to increase scaffold stiffness. Fibronectin was added to formed scaffolds to add a specific ligand binding site for cell migration. Human umbilical vein endothelial cells (ECs) were cultured on glass for 1 day. After which, uv light sterilized scaffolds were placed on top of adherent ECs. EC viability, density and morphology on scaffolds and bare glass were investigated for 9 days after scaffold placement. ECs were found within all uv sterilized scaffolds after 1 day in culture. On pure cellulose acetate scaffolds, EC density plateaued on day 4 at a value approximately 50% of the cell density on paired glass. Cell density on scaffolds with chitosan or fibronectin increased continually over the 10 day culture, surpassing EC density on paired glass. Cell viability was high on all scaffolds and glass (>90%). With increased time in culture, the percentage of elongated ECs increased significantly on all scaffolds. ECs can migrate onto electrospun cellulose acetate scaffolds, showing a preference for a three dimensional matrix over two dimensional glass. Addition of chitosan or fibronectin to electrospun cellulose acetate scaffolds enhanced migration against gravity onto scaffolds and EC elongated morphology. Thanks to NIH HL55492.
Multi‐scale modeling of neuronal adaptation mediated by angiotensin II in the central regulation of blood pressure
Authors: Vadigepalli Rajanikanth , Nikolaev Evgeni , Miller Gregory M , Ogunnaike Babatunde , Gonye Gregory , Kholodenko Boris , Schwaber James S
Published: June 2021

In this study, we focus on the modulation of brainstem cardiorespiratory neurons via angiotensin II receptor, type 1 (AT1R) activation, leading to short‐term consequences on the electrophysiology, and long‐term effects on gene expression. Disregulation of these processes in CNS neurons contributes to several diseases involving homeostasis, most notably hypertension. Our approach is to develop and analyze a multi‐scale mathematical model integrating signaling, gene regulation and electrophysiology in order to identify key mechanisms playing a role in the immediate changes in excitability as well as those involved in long lasting effects on synthesis and release of norepinephrine (NE), a key neurotransmitter involved in cardiovascular regulation. Our model includes detailed kinetics of Gq, PKC, MAPK and PI3K pathways, activation of an immediate early transcription factor AP‐1, and a Hodgkin‐Huxley like description of electrophysiology. Analysis of the model structure and dynamics revealed contributions of multiple mechanisms for activation of AP‐1 leading to novel hypotheses on a dynamic balance between c‐fos and c‐jun activation as an alternative mechanism for transcriptional regulation of Tyrosine Hydroxylase, a rate‐limiting enzyme in the synthesis of NE. Research Support: NIH/HLB R33 HL087361 to JSS and BK.
ADP and CCCP ‐induced increases in mitochondrial free Ca2+: greater contribution of matrix Ca2+ buffering by ATP/ADP
Authors: Haumann Johan , Patel Runa , Aldakkak Mohammed , Rhodes Samhita S , Vinnakota Kalyan C , Dash Ranjan K , Camara Amadou KS , Beard Daniel A , Stowe David F
Published: June 2021

Mitochondrial free calcium (mfCa2+) is a key regulator of bioenergetics, but mfCa2+ is also affected by matrix Ca2+ buffering capacity. We tested how changes in matrix pH (pHm) and ATP/ADP affect mfCa2+. We measured mfCa2+ (indo‐1), pHm (BCECF), membrane potential (ΔΨm)(rhodamine‐123) and redox state (NADH) using spectrofluorometry in guinea pig heart mitochondria suspended in Ca2+ free buffer with 2.5 mM EGTA and 0.5 mM pyruvic acid. ADP (250 μM) and CaCl2 (free [Ca2+] of 25–400 nM) were added to infer the dynamics. Ruthenium red (RR) was given to block Ca2+ uniport. Carbonyl cyanide chlorophenylhydrazone (CCCP) was added to abolish the H+ gradient and lower pHm. Fluorescence signals for mfCa2+ were corrected for NADH in energized mitochondria and for any dye‐induced spectral shifts. We found that mfCa2+ increased more with added ADP than CCCP. RR blocked increases in mfCa2+ with added buffer CaCl2, but did not affect increases due to ADP and CCCP. CCCP alone caused a marked lowering of pHm and abolished ΔΨm, while ADP decreased pHm and ΔΨm to a lesser extent. pHm, mfCa2+, and ΔΨm reverted to baseline as ADP was converted to ATP. We conclude the increase in mfCa2+ after adding ADP is due more to decreased buffering of ADP vs. ATP than to matrix acidification by CCCP. Changes in protein buffering and ion exchange activity may also modulate mfCa2+ during alterations in mitochondrial activity.
Electrical depression in the anoxic turtle cortex is mediated by GABA and initiated by O2‐sensitive ROS depletion
Authors: Pamenter Matthew Edward , Zivcovik George , Buck Leslie Thomas
Published: June 2021

Painted turtle brain survives anoxia but the primary protective mechanisms are unknown. The purpose of this study was to determine if GABA receptors and ROS are involved in the electrical depression observed in anoxic turtle cortex. Using patch clamp techniques electrical activity was recorded form turtle cortical pyramidal neurons undergoing normoxic to anoxic transitions with a ROS scavenging compound (MPG) and GABA receptor manipulation. Anoxia, GABA or MPG increased whole cell conductance (Gw), raised AP threshold (APth) and decreased AP frequency (APf). Simultaneous perfusion of anoxic saline with GABA or MPG, or of normoxic saline with GABA + MPG had no synergistic effects. Blockade of GABAA but not GABAB receptors during anoxia prevented changes in Gw, APth and APf, and the neurons underwent seizure‐like activity and subsequent cell death. Blocking APs with TTX prevented seizure‐like activity and reduced cell death. We conclude that decreased ROS generation during anoxia activates GABAA receptors and increases Cl−conductance in a shunt‐like fashion. This opposes AP generation and reduces the excitability of the cell, reducing the workload of ion pumps and preserving ATP supplies. GABA‐mediated electrical depression appears to be critical to the survival of turtle cortical neurons during anoxia and ROS production may serve as an O2 sensor to mediate this system.
Renin Inhibition Attenuates Ang II Induced Oxidative Stress and Remodeling in the Pancreas of the Ren2 Rat (tg (mREN2)27)
Authors: Habibi Javad , Hayden Melvin R , DeMarco Vincent G , Whaley‐Connell Adam , Schneider Rebecca I , Ferrario Carlos M , Sowers James R
Published: June 2021

The Ren2 rat expresses the mouse renin gene in numerous extra‐renal sites leading to higher tissue Ang II, oxidative stress, insulin resistance, hypertension, and tissue remodeling. Recently, we showed reversal of oxidative stress, myocardial, renal, and vascular remodeling, and skeletal muscle insulin resistance in the Ren2 after treatment with an AT1R blocker or antioxidant. Here we test whether renin inhibition reverses oxidative stress and remodeling in the pancreas of Ren2. Pancreas was harvested from 9 week old male Ren2 and SD rats following 21 day treatment with 50mg/kg/day aliskiren or placebo. Paraffin sections were incubated with primary antibodies to the NADPH oxidase subunits, NOX2 and Rac1, 3‐nitrotyrosine, complex IV subunit I (mitochondrial marker), and secondary anybodies. Fluorescence of laser confocal images was quantified. VVG stained sections were evaluated for periarterial fibrosis. Signal intensities of Rac1 and 3‐nitrotyrosine, but not NOX2, were elevated in both islets and parenchyma of control Ren2 and aliskiren blunted these changes. Arterial fibrosis found in untreated Ren2 pancreas was prevented by aliskiren. Mitochondrial number, which was higher in control Ren2 pancreas, was lowered by aliskiren. The Ren2 pancreas exhibits oxidative stress and remodeling which are reversed by treatment with aliskiren.

Supported by NIH R01‐HL‐63904 and VA Merit (JRS).
Pulmonary arterial oxidative stress in equine recurrent airway obstruction
Authors: Venugopal Changaram , Mariappan Nithya , Elks Carrie , Francis Joseph
Published: June 2021

Oxidative stress in an important characteristic of hyperactive airways diseases such as recurrent airway obstruction (RAO) in horses. The primary source of oxidative stress in RAO is from the elevated amounts of reactive oxygen species (ROS) liberated from the inflammatory cells. ROS alters signal transduction mechanisms, activate redox‐sensitive transcription factors and regulate pro‐inflammatory gene expressions. Thus, ROS levels have important consequences for pathogenesis, severity and therapeutic management of RAO. The involvement of Tumor Necrosis Factor‐alpha (TNFα), a cytokine, is well established in inflammation. Therefore, the objective of the study was to determine oxidative stress in pulmonary arterial rings of RAO‐affected and unaffected horses and to test the effect of TNFα blockade using pentoxifylline. Arterial rings were collected from right diaphragmatic lung lobe and the ROS released by the tissues were measured using Electron Paramagnetic Resonance (EPR). The results showed that total ROS was significantly greater in RAO affected than the unaffected horses. In addition, incubation of the arterial rings with pentoxifylline reduced the ROS level in the RAO horses. The study suggested that pentoxifylline could be effective in allevieating oxidative stress in RAO. Supported by Equine Health Studies Program of LSU School of Veterinary Medicine.
Antioxidants Increase Tissue Plasminogen Activator (tPA) Synthesis in Human Mesothelial Cells via MAPK Activation Signals
Authors: Reed Karen L , Gower Adam C , Heydrick Stanley J , Stucchi Arthur F , Becker James M
Published: June 2021

Peritoneal adhesions, a major complication of abdominal surgery, are due, in part, to reduced levels of postoperative peritoneal tPA, an enzyme that facilitates degradation of fibrinous adhesions. Mesothelial cells lining the peritoneum are a primary source of tPA. Since the antioxidants vitamin E and methylene blue (MB) reduce adhesion formation in animal models, the aim of this study was to determine their effects on tPA synthesis in LP‐9 cells, a non‐transformed human mesothelial cell line. Methods: Cells were incubated with the vitamin E analog trolox (.05, .5, 5mM) or MB (.025, .25, 2.5, 25μM) for 24 hrs. To examine signaling pathways, trolox (5 mM) and MB (25 μM) were administered with inhibitors of transcription (2μM actinomycin D), translation (50μM cyclohexamide), and the MAP kinases p38 (10μM SB202190), JNK (10 μM Inhibitor II) and ERK1/2 (10μM PD98059). Culture media (CM) was analyzed for tPA content by ELISA. Results: Trolox and MB at the highest doses increased (p<0.05) tPA in CM by 464 ± 97% and 437 ± 57 %, respectively, compared to untreated cells (100 ± 5.6%). Actinomycin D, cyclohexamide and the JNK inhibitor blocked this response while the ERK1/2 inhibitor had no effect. The p38 inhibitor increased tPA content in CM to 208 ± 30 % (p<0.05) of control, however, it blocked the trolox and MB response to the level of the inhibitor alone. Conclusion: Trolox and MB increase tPA synthesis in LP‐9 cells, in part, via the p38 and JNK signaling pathways. These in vitro data suggest that, in vivo, mesothelial cell synthesis of tPA may be regulated by the oxidant status of the peritoneum. Supported by the Smithwick Endowment from the Dept of Surgery at BUSM.
670 nm LED Treatment Affects Hyperglycemia‐Induced Oxidative Stress in Skeletal Muscle in Diabetic Rats
Authors: Sandoval Christopher L , Wilson Robert W , Gopalakrishnan Sandeep , Lark Daniel S , Eells Janis T , Snyder Ann C
Published: June 2021

Oxidative stress is important in the pathogenesis of diabetes‐induced muscle atrophy. Low energy photon irradiation with 670 nm light has been shown to attenuate oxidative stress following ischemia‐reperfusion injury in the heart. PURPOSE: To determine if 670 nm LED treatment (NIR) would attenuate oxidative damage in a model of type I diabetes, the streptozotocin (STZ)‐treated rat. METHODS: Wistar rats were divided into three treatment groups: Control (CON), STZ‐treated (STZ) and 670 nm LED treated STZ (NIR‐STZ). Diabetes was induced by STZ (50 mg/kg, ip). NIR‐STZ animals were treated once per day for 5 min for 17 days with 670 nm LED arrays (6 joules/cm2; QDI, Barneveld, WI). Animals were euthanized on day 18 and the rectus femoris (RF) muscle was dissected and flash frozen for biochemical analysis of lipid peroxidation. RESULTS: Lipid peroxidation in the RF was slightly increased in the STZ and NIR‐STZ groups relative to control. Total antioxidant capacity in the RF was increased in STZ and NIR‐STZ rats when compared to that of the CON. CONCLUSIONS: Hyperglycemia increased the production of reactive oxygen species. In the acute stage of diabetes, antioxidant proteins are upregulated in response to oxidative stress and protect against lipid peroxidation. Diabetic animals treated with 670 nm light exhibited an additional increase in antioxidant capacity. Funded by: Biomedical Technology Alliance Award and UWM RGI.
Role of the AE3 Chloride/Bicarbonate Exchanger in the Development of Cardiac Hypertrophy
Authors: Sowah Daniel , Alvarez Bernardo , Shull Gary , Casey Joseph
Published: June 2021

Cardiac hypertrophy is one of the determinants of heart failure, the leading cause of hospitalization in the elderly in Western societies. Several factors contribute to the development of cardiac hypertrophy. Emerging evidence suggest that stimulation of the hypertrophic transport metabolon, a complex involving the Na+/H+ exchanger, NHE1, carbonic anhydrase II and the AE3 Cl−/HCO3− exchanger, plays a key role in the development of hypertrophy. To understand the contribution of AE3 to the hypertrophic cascade, knock‐out mice lacking the gene that encodes AE3 (AE3−/−) were generated. The body weights showed no significant differences between the wild type (AE3+/+), the heterozygous (AE3+/–) and the knock‐out (AE3−/−) littermates. Interestingly, the heart weight to body weight ratio, an index of cardiac hypertrophy, was markedly reduced in the AE3−/− mice (82 ± 2% vs. AE3+/+); the AE3+/− mice were identical to the AE3+/+ mice (102 ± 3% vs. AE3+/+). Phenylephrine (PE), 10 μM, or angiotensin II (ANG II), 1 μM, added 18 h following culturing of adult cardiomyocytes and present for 24 h, increased the cell size of isolated cardiomyocytes of AE3+/+ mice by 20 ± 2% but AE3−/− cardiomyocytes were unresponsive to PE‐ or ANG II‐induced hypertrophy (101 ± 4% vs. AE3+/+). Our data demonstrate that deletion of the AE3 Cl−/HCO3− exchanger gene renders the myocardium significantly less sensitive to cardiomyocyte hypertrophy.

Supported by the Heart and Stroke Foundation of Alberta.
Modulation of proton ATPase activity in MDCK ‐ C11 cells
Authors: Perez Ricardo Fernandez , Tararthuch Ana Lucia , Santos Priscilla Marys Dos
Published: June 2021

The objective was to characterize the biochemical activity of the proton pumps present in the MDCK ‐ C11 cells as well as to identify several additional mechanisms of their regulation. We used cells homogenates to determined H+ ATPases activity. Changes in [Ca2+]i in the cells was determined using the Ca2+ ‐ sensing dye Fluo‐4. MDCK C11 cells present a bafilomycin sensitive (H+ ATPase), and a vanadate sensitive (H+‐K+ ATPase) activity. The H+ ATPase showed a pH optimum of 6.05 when assayed in the absence of K+. This pH optimum is close to that obtained in plasma membranes from intercalated cells. ATPase activity was also stimulated in a dose‐dependent fashion as K+ concentration was increased between 0 and 50mM. The Lineweaver‐Burke plot of K+ dependent ATPase activity indicates apparent Km of 0.13mM and Vmax of 22.01nmol Pi.mg−1.min−1. Incubation of cells monolayers with 10−8M aldosterone during 24h increased significantly H+ ATPase activity (6.61±0.54 vs 4.37±0.68nmol Pi.mg−1.min−1 basal activity, p<0.05), but not altered H+‐K+ ATPase activity. This effect on H+ ATPase activity was prevented by 10−5M spironolactone and cycloheximide (10−5M). Incubation of cells monolayers with aldosterone for only 1h not altered H+ ATPases activity. H+ ATPase activity was also stimulated by 10−11M vasopressin (7.93±0.55 vs 4.86±0.50nmol Pi.mg−1.min−1 basal activity, n=17, p<0.01), an effect prevented by a V1‐receptor‐specific antagonist. On the other hand, a pharmacological level of AVP (10−9M) didn't modified significantly this activity. Both doses increased significantly [Ca2+]i: 47.41±3,70% (n=5) if compared with control levels with 10−11M, and 324.99±32.42% with 10−9M. In summary, homogenates of MDCK ‐ C11 cells present both a H+ ATPase and a H+‐K+ ATPase activities; and the H+ ATPase activity is activated by aldosterone through a genomic pathway and by vasopressin through V1 receptors.
Mild Renal Insufficiency Results in Increases in Myocardial Collagen: Evidence for a Heart – Kidney Connection
Authors: Martin Fernando L. , Huntley Brenda K. , Harders Gerald E. , Chen Horng H. , Cataliotti Alessandro , Burnett John C.
Published: June 2021

Decreased renal function is associated with increased CV morbidity and mortality. The changes mild renal impairment may cause structurally/molecularly in the heart remain unclear. We hypothesized that mild renal dysfunction produced by unilateral nephrectomy(UNX)results in changes in ventricular structure and gene expression in the absence of hypertension or vol overload. Wistar rats were used[sham (S; n=10) and UNX; n=9]. At 4 weeks, GFR was determined by inulin clearance and RBF by PAH. Hearts and kidneys were harvested for histological analysis. Cardiac function assessed by echo. Genomewide microarray analysis was performed on LV myocardium (Affymetrix). Glomerular hypertrophy was observed in UNX. GFR tended to decrease with a reduction in RBF (S:8±1, Nx:5±1 ml/min, p<0.005). Blood pressure (BP), Na and water excretions were maintained without activation of PRA, aldo or BNP. LVEF and LV diameters were normal. Microarray analysis of LV revealed that 278 genes changed with UNX (1.5 fold, P<0.05) related to cell growth, bone remodeling and muscle contraction (Z value>2). Importantly, Pic Red staining in the LV of UNX revealed greater fibrosis (S:2±0.1, Nx:4±0.4 %, p<0.001). We conclude that mild renal insufficiency initiates myocardial gene responses and fibrosis independent of alterations in the circulating RAAS, BNP, BP, vol overload or systolic dysfunction.
Macula densa cells detect altered tissue metabolism via succinate and GPR91
Authors: Vargas Sarah Laurin , Toma Ildiko , Kang Jung Julie , Hanner Fiona , Peti‐Peterdi Janos
Published: June 2021

Cells of the macula densa (MD) can sense changes in tubular fluid composition and regulate glomerular filtration rate and renin release from the juxtaglomerular apparatus (JGA). MD control of renin release and renin‐angiotensin system (RAS) activation involve MD p38, ERK1/2, COX‐2, synthesis and release of PGE2 acting on JG cells. Diabetes mellitus (DM) is characterized by MD p38 and RAS activation. We hypothesized that MD cells sense a tubular metabolic factor in DM which directly triggers renin release. Using RT‐PCR and immunohistochemistry (IHC), GPR91, a novel metabolic receptor activated by the Krebs‐cycle intermediate succinate, was detected in the mouse MD cell line MMDD1and at the apical plasma membrane of MD cells. Succinate caused a rapid activation of p38and ERK1/2 in MMDD1 cells within 7.5 min detected by immunoblot, which was abolished by GPR91 siRNA. Renal cortical pp38 and renin content increased 2‐fold in STZ‐DM GPR91+/+, but not in GPR91−/− mice. IHC also confirmed the abundance of COX‐2 in the MD in STZ‐DM which was absent in GPR91−/− mice. Confocal microscopy utilizing a PGE2 biosensor technique detected succinate‐induced PGE2 release from MMDD1 cells. In summary, the metabolic receptor GPR91 is expressed in the MD apical membrane and activates p38 and ERK1/2. MD GPR91 activation, most likely through succinate accumulation in the tubular fluid is involved in renin signaling and RAS activation in early DM.