bluuebunny/crossref_metadata_2025 huggingface dataset:
Local control model illustrates how action potential morphology affects Ca2+ release
Authors: Gauthier Laura Doyle , Greenstein Joseph L , Winslow Raimond L
Published: June 2021

The local control theory of excitation‐contraction (EC) coupling states that regulation of calcium (Ca2+) release occurs at the nanodomain level, where openings of single L‐type Ca2+ channels (LCCs) trigger openings of small clusters of ryanodine receptors (RyRs) co‐localized within the dyad. We have formulated a deterministic “coupled LCC‐RyR model” that captures the local control scheme's voltage‐dependent gain and graded release properties. The model presented here combines our local control model with a prior model of guinea pig myocyte electrophysiology, metabolism, and isometric force production. It predicts a causal relation between action potential (AP) shape and Ca2+ release timing. In species expressing the transient outward current (Ito), APs exhibit a “notch” and Ca2+ transients peak soon after the AP upstroke. However guinea pig lacks Ito, and its Ca2+ transients peak later in the AP plateau. Here the late Ca2+ transient peak arises from low initial EC coupling gain at the AP peak. Gain increases gradually during plateau repolarization. Addition of Ito and thus the AP notch leads to higher gain early in the AP, causing earlier Ca2+ transient peaks. Our model predicts that AP shape differences due to current expression heterogeneity will impact the temporal waveforms of Ca2+ and force transients and will influence tissue‐level electrical propagation. Funded by NIH RO1HL1052160 and NSF GRFP.
Uridine adenosine tetraphosphate (Up4A) as a novel coronary vasodilator in health and disease: Role of purinergic P1 and P2 receptors
Authors: Zhou Zhichao , Duncker Dirk Jan , Merkus Daphne
Published: June 2021

Up4A is a potent endothelium‐derived contracting factor that contains purine and pyrimidine moieties and activates purinergic P2X and P2Y receptors. Up4A has recently been found to also have vasodilator effects. Here, we evaluated Up4A in isolated coronary small arteries from normal (N) swine and swine after myocardial infarction (MI), and studied the purinergic receptor subtypes involved. Surprisingly, Up4A (10−9–10−5 M) failed to induce contraction in small arteries from N and MI, but produced dose‐dependent relaxation in vessels from N and MI preconstricted with U46619. Sensitivity to Up4A was lower in MI than N. In N, Up4A induced vasodilation was reduced by nitric oxide (NO) inhibition with L‐NAME to a similar extent as by nonselective P1 antagonist 8PT and A2A antagonist SCH58261, while nonselective P2 antagonist PPADS, P2X1 antagonist MRS2159, and P2Y1 antagonist MRS2179 had no effect. In MI, dilation by Up4A was blunted by P2‐ (PPADS, MRS2159, MRS2179) and P1‐inhibition (8PT, SCH58261) and L‐NAME. Q‐PCR confirmed the presence of coronary P2X1, P2Y1 and A2A receptors. Thus, Up4A exerts a dilator rather than a constrictor effect in coronary microvessels which in N occurs via P1 (particularly A2A) mediated activation of NO, and both P1 and P2 in MI. These findings indicate that the mechanism of vasodilation by Up4A is different in the diseased heart.

Supported by China Scholarship Council (2009624027)
Calcitonin Gene Related Peptide Is Not Required for the Pressor Response to Water
Authors: Mai Tu , McHugh Julia , Appalsami Martin , Supowit Scott , Dipette Donald , Gangula Pandu , Diedrich Andre’ M. , Robertson David
Published: June 2021

Water ingestion induces a robust increase in blood pressure in patients with baroreflex impairment. To better understand this phenomenon, we use a modified sino‐aortic denervated mouse model. Water (750μL/30g body weight), but not saline solution, infused into the duodenum of these mice stimulates an increase in blood pressure (BP) similar in magnitude and time course to that in patients. This indicates that hypo‐osmolality is the stimulus for this response. Previous studies provided evidence that increased sympathetic outflow underlies this pressor effect. However, physiological and molecular mediators of this effect remain unknown. Calcitonin Gene Related Peptide (CGRP) exerts effects centrally and peripherally in the gastrointestinal, respiratory and endocrine systems. It has been implicated in conveying afferent information that leads to sympathetic activation in response to sensory input. To determine whether CGRP plays a crucial role in the osmopressor response, water was given intraduodenally to sino‐aortic denervated α‐CGRP −/−mice. Although CGRP−/− mice had higher baseline BP before denervation (MAP = 82 ± 2 mmHg in CGRP−/− mice compared to 77 ± 2 mmHg in the WT, P value<0.001), the magnitude of BP increase after water infusion was similar in the knockout (ΔMAP= 14.4 ± 5 mmHg) and wild type mice (ΔMAP= 15.9 ± 4 mmHg) (P value = 0.49). This indicates that CGRP is not required for the pressor effect of water.
Blood pressure profile and response to NG ‐nitro‐L‐arginine methyl ester challenge in conscious TRPV4‐deficient mice
Authors: Nishijima Yoshinori , Lund Hayley , Zheng Xiaodong , Mattson David , Zhang David
Published: June 2021

Endothelial TRPV4 is a Ca2+ entry channel critically involved in endothelium‐mediated dilation in both conduit and resistance vascular beds. However the importance of this TRPV4‐mediated vasodilation in vivo has not been well characterized. We investigated the role of TRPV4 in blood pressure (BP) regulation under normal conditions and in response to a hypertensive challenge. The study was performed in TRPV4‐deficient (TRPV4−/− ) and wild‐type (WT) mice implanted with a radiotelemetry device for continuous recording of mean arterial pressure (MAP), heart rate (HR) and activity. Basal MAP tends to be lower in TRPV4−/− mice (101 ± 3 mmHg, n=3, p=0.054) than in WT controls (108 ± 1 mmHg, n=4), whereas HR was comparable in TRPV4−/− (565 ± 11 bpm) and WT mice (571 ± 8 bpm). Hypertension was induced by administration of L‐NAME (0.5 g/L), a nitric oxide synthase inhibitor, in drinking water for 7 days. The L‐NAME‐induced increase in MAP was similar in TRPV4−/− (9.8 ± 1.6 %) and WT (9.5 ± 1.6 %), although the concomitant reduction in HR was less in TRPV4−/− (−7.5 ± 1.1 %) than in WT (−12.6 ± 1.9 %). The activity was lower in TRPV4−/− (4.1 ± 0.8 units) compared with WT (6.3 ± 1.5 units). In conclusion, TRPV4−/− mice exhibit a trend toward a lower basal BP but retain a normal BP response to an L‐NAME challenge. These findings call for further evaluation of the significance of TRPV4 in the regulation of vascular tone in vivo.
Endothelial Focal adhesion kinase maintains lung fluid balance and prevents cytokine storm
Authors: Thennes Tracy , Tauseef Mohammad , Bonini Marcelo , Gothert Joachim , Shen Tang-Long , Guan Jun-Lin , Sadikot Ruxana , Mehta Dolly
Published: June 2021

Sepsis‐induced acute lung injury (ALI) results from inflammatory cell infiltration and protein‐rich edema formation. Since focal adhesion kinase (FAK) influences adherens junction formation required for establishing the endothelial barrier, we generated tamoxifen‐inducible endothelial (EC) FAK deficient transgenic mice (EC‐FAK−/−) to address whether FAK controls pulmonary fluid balance and inflammation. EC‐FAK deletion severely disrupts the lung vasculature and promotes pro‐inflammatory cytokine generation such that EC‐FAK−/− mice have increased mortality to endotoxin (LPS). FAK null lung ECs showed actin stress fiber formation, adherens junction disassembly and increased pro‐inflammatory cytokine levels, naming EC‐FAK as a central regulator of lung fluid balance and inflammation. Mechanistically, loss of FAK markedly altered the balance of small GTPases RhoA and Rac1; RhoA activity was significantly increased whereas Rac1 was decreased in FAK null ECs. FAK deletion also amplified p38MAPK activity, which is known to generate cytokines downstream of toll‐like receptors. We confirmed these findings using siRNA‐mediated FAK suppression in human pulmonary ECs. Moreover, EC‐FAK levels were decreased in lungs of patients diagnosed with lung disease and in mice following sepsis. Thus, upregulating endothelial‐FAK provides an unexpected mechanism for preventing cytokine storm and ALI.
RAGE signaling influences tobacco smoke‐induced inflammation by pulmonary macrophages
Authors: Robinson Adam B. , Johnson KacyAnn D. , Bennion Brock G. , Reynolds Paul R.
Published: June 2021

Receptors for advanced glycation end‐products (RAGE) are multi‐ligand cell surface receptors expressed by lung cells and expression increases following exposure to cigarette smoke (CS). The current study sought to characterize the pro‐inflammatory effects of RAGE in pulmonary macrophages (PMs) following CS exposure. Acute exposure to CS via nasal instillation revealed diminished bronchoalveolar lavage (BAL) cellularity and fewer macrophages in RAGE null mice compared to controls. Primary PMs were obtained from BAL, exposed to CS in vitro, and analyzed. CS significantly increased RAGE expression in wild type PMs. By ELISA, wild type PMs increased active Ras, a small GTPase that perpetuates pro‐inflammatory signaling, following CS exposure. Conversely, RAGE null PMs had less Ras activation compared to wild type PMs after exposure to CS. Assessment of signaling intermediates including p38 and NF‐kB revealed CS‐induced inflammation may occur in part via RAGE signaling. Lastly, qRT‐PCR and ELISA revealed that secretion of cytokines such as TNF‐α and IL‐1β were decreased in RAGE null PMs exposed to CS compared to CS‐exposed wild type PMs. These results show for the first time that primary PMs orchestrate CS‐induced inflammation, at least in part, via RAGE‐mediated mechanisms. This work was supported by the Flight Attendant's Medical Research Institute (FAMRI, PRR) and a BYU Mentoring Environment Grant (PRR).
Tauroursodeoxycholic acid (TUDCA) improves function in cold storage hearts
Authors: Porter Karen , Mingo Tyler , Garry Glynnis A , Garry Mary G
Published: June 2021

Bile acids have been used to improve various liver conditions and are now being investigated as putative therapies for neurological and cardiovascular pathologies. Tauroursodexycholic acid (TUDCA), a taurine conjugate of ursodeoxycholic acid (UDCA), has been demonstrated to reduce scar size and apoptosis resulting from myocardial infarction. Since TUDCA has been shown to protect against acute tissue injury, we hypothesized that addition of TUDCA to the cold storage solution, University of Wisconsin (UW), will improve heart function during reperfusion and lengthen the time the heart can be stored. Male rat hearts were explanted and perfused with either UW alone or UW+TUDCA (1 mM) then stored in the solution for 4 hours at 4°C. Following cold storage the hearts were reperfused with Krebs‐Henseleit buffer via a Langendorff isolated heart apparatus to measure cardiovascular function. Hearts stored in UW+TUDCA demonstrated increased left ventricular pressure (39.4 mmHg vs. 27.1 mmHg respectively) and developed pressure (90.8 vs. 64.4 mmHg respectively) compared to UW alone. Additionally, amount of time for relaxation of the heart was increased in the UW‐TUDCA group (−1648.9 mmHg/s vs. −1226.7 mmHg/s) demonstrating improvement in the heart function. These data indicate that bile acids may be beneficial in reducing tissue injury and increasing function of explanted hearts.

Studies supported by AHC seed grant.
Spatio‐temporal patterns of apoptotsis and mitosis during organismal regeneration in a basal chordate
Authors: Gilman Joshua P , Lauzon Robert J
Published: June 2021

Ascidians are marine invertebrate chordates that are closely related to vertebrates. The colonial ascidian Botryllus schlosseri represents a model system for the study of homeostasis and regeneration. During its weekly budding cycle known as blastogenesis, new buds are generated by asexual reproduction, and as they reach functional maturity, replace the old asexual generation of adult zooids. During this phase of blastogenesis (called takeover), the entire generation of adult zooids undergoes apoptotic programmed cell death (PCD) followed by its resorption and clearance by circulating phagocytes within 24–36 hours. The goal of the present study was to investigate the spatio‐temporal relationship between PCD and mitosis using polyester wax‐embedded serial sections from colonies at various phases of the blastogenetic cycle. We have used an immunofluorescence microscopy approach in conjunction with the use of an antibody against phospho histone H3 (pHH3) to detect mitotic cells. Here, we demonstrate that mitosis is largely restricted to tissues and organs of developing buds as well as male germline cells of both buds and adult zooids. We are currently using pHH3 immunoreactivity simultaneously with terminal deoxynucleotidyl transferase dUTP end‐labeling (TUNEL assay) to respectively visualize mitotic and apoptotic cells respectively. Experiments are currently underway to determine how these processes quantitatively differ during growth and death phases of a B. schlosseri colony.
Artificial Selection for High Aerobic Capacity is Protective against Weight Gain via Thermogenesis?
Authors: Torvinen Sira Maria , Silvennoinen Mika , Mikkonen Maria , Koch Lauren G. , Britton Steven L. , Kainulainen Heikki
Published: June 2021

Higher spontaneous activity and non‐exercise activity thermogenesis (NEAT) contribute significantly to daily energy balance, body weight, and therefore in health. It was previously shown that, relative to body mass, rats selectively bred for high intrinsic aerobic capacity (HCR = high‐capacity runner) consume more energy and are more spontaneously active than low‐capacity runner (LCR) counterparts. We tested whether HCRs have higher rectal temperatures after 2 h fasting and during glucose tolerance test compared to LCRs. We studied 40 HCR/LCR female rats for the fasting condition, and 20 HCR/LCR female rats for the glucose tolerance test. Rats were from 27th generation of selection and aged 9 months. During the glucose tolerance test we controlled the spontaneous activity levels of the rats with ground reaction force recording. Our study revealed that after 2 h fasting HCRs had significantly higher rectal temperature compared to LCRs (p < 0.05). During glucose tolerance test the rectal temperature of HCRs was significantly higher compared to LCRs throughout the test protocol (p < 0.05). In the present study the spontaneous activity of HCRs was higher at the beginning (p < 0.05), but not at the end of the glucose tolerance test, suggesting that higher activity of HCR rats does not exclusively explain higher NEAT. This study was supported by the Ministry of Education (Finland) and the National Institute of Health.
Effect of hypoxia on single skeletal muscle fiber contractility at physiological temperature
Authors: Shiah Amy Anling , Nogueira Leonardo , Gandra Paulo Guimaraes , Hogan Michael C.
Published: June 2021

Hypoxia has been associated with an increased reactive oxygen species (ROS) production in contracting skeletal muscle. However, it is not known whether hypoxia can modulate the muscle contractility, particularly at physiological temperature. We hypothesized that a decrease in intracellular PO2 would regulate force in skeletal muscle through ROS. Intact single fast‐twitch fibers from mouse muscle (n=20) were stimulated (1–200 Hz) at ambient PO2 (~150 Torr) and under hypoxia (2–4 Torr) at 22, 32 and 35ºC. At both 22 and 32ºC, hypoxia did not affect contractility. However, at 35ºC under hypoxia, there was a significant (P<0.05) decrease in submaximal tension (28 ± 11%) compared to ambient PO2. Treatment of single myofibers with tiron (1 mM) and N‐acetyl‐cysteine (NAC; 0.5 mM) at low PO2 did not alter the decrease in submaximal force seen during hypoxia. However, with H2O2 (100 μM), submaximal tension increased after 2–5 min (51 ± 4%), and then decreased thereafter. These results suggest that hypoxia at physiological temperature diminishes submaximal force in single fast‐twitch fibers, but not in a ROS dependent manner. Supported by NIH (AR040155‐17) and APS Undergraduate Summer Fellowship.
Integrative physiology of antidepressant drug action
Authors: Lowry Christopher Alan , Hale Matthew William , Dady Kathleen Francis , Lukkes Jodi L , Kelly Kyle John , Raison Charles L
Published: June 2021

Previous studies have demonstrated that depressed patients have dysfunction of thermoregulatory cooling, while antidepressants of diverse pharmacological profiles induce sweating as a clinical side effect, suggesting that the pathophysiology of depression as well as antidepressant drug action may involve interactions with thermoregulatory pathways. To test this hypothesis, we investigated interactions between acute whole body warming and treatment with the selective serotonin reuptake inhibitor, citalopram, on body temperature, measured using chronically implanted telemetric recording devices, and antidepressant‐like behavior in the forced swim test in rats. Using a subthreshold dose of citalopram, we found that citalopram, by itself, induced hyperthermia that was comparable to that induced by exposure to increased ambient temperature (37 degrees C) for 85 min. Neither citalopram by itself, nor exposure to increased ambient temperature induced antidepressant‐like effects in the forced swim test. However, when rats were both treated with citalopram and exposed to elevated ambient temperature, rats experienced an exaggerated hyperthermia that was highly correlated with antidepressant‐like behavioral responses. These data provide a rationale for novel therapeutic strategies for the treatment of affective disorders, including development of novel antidepressant drugs.
Release of acetylcholine during whole‐body heating in aged skin
Authors: Shibasaki Manabu , Negishi Hiroko , Kubo Hiroko , Okazaki Kazunobu , Crandall Craig G
Published: June 2021

Sweat rate (SR) to a whole‐body heat stress is attenuated in aged skin. Possible mechanisms of this attenuation are 1) reduced neural signaling, 2) attenuated release of neurotransmitter, and/or 3) decreased sweat gland responsiveness to acetylcholine (ACh). We tested the hypothesis that attenuated release of ACh in aged skin may contribute to decreases in sweating during a heat stress. Intradermal microdialysis probes were placed in dorsal forearm and lower leg skin in 8 young (22±1 yrs) and 7 old (70±1 yrs) healthy subjects. Local SR was measured over the microdialysis membranes by the ventilated capsule method. Ringerˈs solution containing 10mM of the acetylcholinesterase inhibitor neostigmine was perfused through each membrane (perfusion rate: 2 μl/min) and the effluent was collected at 8 min increments. ACh in the effluent was measured by high‐pressure liquid chromatography combined with electrochemical detector. Whole‐body heating increased tympanic temperature ~ 1.2°C. Throughout the heat stress SR was lower in the aged group, whereas the release of ACh was similar between groups at both forearm and leg sites. Therefore the SR/ACh ratio in the 20 μl sample was significantly lower in the aged group (Forearm: 0.10±0.07 vs 0.21±0.11 mg/cm2/min/fmol, Leg: 0.10±0.06 vs 0.25±0.13 mg/cm2/min/fmol, both P<0.05). These results suggest that the age‐related reduction in SR is not due attenuated release of ACh.
The change in lower limb venous compliance is different between women and men following 60 days of head‐down bedrest but is not associated with venoconstrictor dysfunction
Authors: Westby Christian M , Lee Stuart MC , Stenger Michael B. , Platts Steven H.
Published: June 2021

Space flight‐induced orthostatic intolerance (OI) is more prevalent in female (F) than male (M) astronauts. The mechanisms explaining the higher incidence of OI in F are unclear. We tested the hypothesis that venous compliance would be higher in F more than M following 6° head‐down bed rest (BR) and would be associated with constrictor dysfunction. Using 2‐D ultrasound, dorsal hand (DHV) and dorsal foot (DFV) vein compliance were determined in 24 subjects (10 F, 14 M; 35±1 yr) by measuring mean diameter response to increasing congestion pressure (0, 20, 30, and 40 mmHg) before and after 60 days of BR. Constrictor function was assessed by intravenous infusions of Ketorolac (KE; 1.5 μg/min), Phenylephrine (PE; 3160 ng/min), and L‐NMMA (50 μg/min). The effect of BR between F vs. M and hand vs. foot, was determined using mixed‐effects linear regression. DFV but not DHV compliance changed in response to BR (p=0.012). Mean DFV increased significantly (0.903 mm to 1.191 mm) in F but decreased in M (1.353 mm to 1.154 mm). DFV constrictor response was not different between sexes in response to BR (KE; p=0.647, PE; p=0.717, and L‐NMMA; p=0.825). These data suggest that the higher incidence of OI in F astronauts may be related to increased lower limb venous compliance, contributing to blood pooling upon standing. Notably, the differential change to DFV compliance between sexes was not associated with impaired constrictor function.
Anatomical and functional connections between the locus coeruleus (LC) and the nucleus of the solitary tract (NTS) in neonatal rats
Authors: Lopes Luana Tenorio , Patrone Luis Gustavo Alexandre , Li Ke-yong , Imber Ann , Graham Cathy , Gargaglioni Luciane , Putnam Robert
Published: June 2021

To study the communication among chemosensitive brainstem areas, we injected rhodamine beads into the LC in rat pups (P7–P10). After 8–10 days, retrograde labeling was seen in the brainstem, including neurons from many chemosensitive regions, such as contralateral LC, caudal NTS (cNTS), retrotrapezoid nucleus, medullary raphé and periaqueductal gray. Similar connections were seen with cNTS injection, although less labeling was seen in each area. We patched cNTS neurons that project to the LC and found that 4 of 5 retrogradely‐labeled cNTS neurons had increased firing rate (FR) in response to hypercapnic acidosis (HA‐15% CO2), even in synaptic blockade medium (SNB—high Mg2+/low Ca2+). In contrast, 2 of 3 retrogradely‐labeled LC neurons that project to the cNTS had reduced FR in response to HA both in the presence and absence of SNB. We loaded the patched neuron with Lucifer Yellow (LY) from the pipette and verified that the neuron was labeled for both rhodamine and LY. Our studies show: extensive connections among chemosensitive brainstem regions; HA‐induced increased FR of cNTS neurons results in increased action potentials (APs) to the LC; and HA‐induced decreased FR of LC neurons results in reduced APs to cNTS neurons. Complex interactions clearly exist among chemosensitive regions within the central respiratory control network.

Supported by FAPESP, CAPES, CNPq, NIH R01‐HL‐56683 and WSU Research Incentive Grant
Effects on ventilation (VE) and neuromodulator concentration of cholinergic receptor blockade at the pre‐Bötzinger Complex (preBötC)
Authors: Muere Clarissa , Miller Justin , Neumueller Suzanne , Mouradian Gary , Pan Lawrence , Hodges Matthew , Forster Hubert
Published: June 2021

We previously found that microdialysis of the muscarinic acetylcholine receptor antagonist, Atropine (AT, 50mM) in mock cerebrospinal fluid (mCSF) into the Kӧlliker‐Fuse Nucleus (KFN) of awake and sleeping goats (n=6) reduces VE and breathing frequency (f) (J Appl Physiol, 109: 159–170, 2010). In contrast, dialysis of 50mM AT into the preBötC of awake goats (n=6) increased VE and f, and increased the concentration of serotonin (5‐HT) and substance P (SP) in the effluent mCSF (FASEB J, 25: 1074.1, 2011). To gain insight into the mechanism by which cholinergic receptor blockade in the preBötC increases VE and f, we dialyzed in one awake goat, mCSF into the preBötC on one side, and simultaneously dialyzed 5mM or 50mM AT into the preBötC of the contralateral side. 5mM AT did not affect VE, f, or effluent 5‐HT, but minimally increased effluent SP, indicating a dose‐dependent AT effect. In contrast, 50mM AT increased VE, f, and effluent 5‐HT and SP. The increase in 5‐HT and SP was restricted to the side receiving AT. These data suggest that cholinergic receptor blockade in the preBötC affects local, not global, levels of these excitatory neuromodulators, which affect the level of breathing. We hypothesize that AT blocks cholinergic receptors of pre‐synaptic 5‐HT and SP terminals at the preBötC, the effect of which is enhanced release of 5‐HT and SP, which subsequently increases VE and f. Support: NIH HL25739 & Veterans Administration
Partial silencing of medullary serotonin (5HT) vesicular neurotransmission inhibits respiratory long term facilitation (LTF) but does not prevent apneas in mice
Authors: Barrett Karlene T , Brust Rachael , Kim Junchul , Dymecki Susan , Li Aihua , Nattie Eugene E
Published: June 2021

LTF, a sustained increase in ventilation (VE) after repeated exposure to intermittent hypoxia, is 5HT dependent and is thought to prevent apneas. The sudden infant death syndrome (SIDS) is associated with 5HT deficiency and some SIDS infants had increased and prolonged apneas prior to death. In this study we used the Pet1:flpe ‘silenced’ mouse to test the hypothesis that partial inhibition of 5HT secretion inhibits LTF and leads to increased apneas in early postnatal life. In these mice, the dual activity of Cre and Flpe recombinase activates expression of tetanus toxin light chain in 5HT neurons, which cleaves the VAMP2 protein that anchors synaptic vesicles to the synaptic membrane, thereby inhibiting synaptic vesicular secretion in these neurons. In ‘silenced’ and control pups at postnatal days (P) 5, 8 and 12, we measured VE before, during and after intermittent hypoxia and apnea frequency in the pre‐ and post‐hypoxic periods. VE was normal in ‘silenced’ pups at baseline, but was 10% greater than controls during hypoxia. During the post‐hypoxic period, control pups at P5 and P8 had a 16% and 26% increase in VE normalized to metabolic rate (VE/VO2) respectively, indicative of LTF but VE/VO2 was unchanged in ‘silenced’ pups. The apnea frequency did not change in either genotype during the post‐hypoxic period, despite LTF in control pups. We conclude that in early postnatal life LTF requires 5HT but does not prevent apneas.
Supplementation of L‐arginine and/or inhibition of arginase enhance the effect of inhaled NO on airway function of hyperoxia‐exposed rat pups
Authors: Zaidi Aliya , Jafri Anjum , Zaidi Syed I. A.
Published: June 2021

Hyperoxia‐induced lung injury in neonates is characterized by both enhanced contraction and impaired relaxation of airway smooth muscle. We have demonstrated that hyperoxia‐induced lung reactivity is due to impaired NO‐mediated signaling. We hypothesize that supplementation of L‐arginine and/or blockade of arginase will enhance the effect of inhaled NO (iNO). The rat pups (P0) were exposed for 7 days to room air (RA), 50% O2, or 50% O2 plus NO (2 or 20 PPM) in presence or absence of L‐arginine and nor‐NOHA administration. 50% O2 significantly (p<0.001) increased the EFS‐induced contraction of tracheal rings as compared to RA exposure. The iNO reversed the hyperoxia‐induced increased contraction of tracheal rings in a dose dependent manner. The reduction effect of iNO was enhanced significantly by arginase inhibition but not by L‐arginine supplementation. The nor‐NOHA in combination with 2 PPM iNO decreased contraction of tracheal rings equal to 20 PPM iNO alone. Moderate hyperoxic exposure significant enhanced arginase I expression in tracheal epithelium. The iNO (20 PPM) has inhibited arginase I expression below RA level in hyperoxic animals. The arginase I expression was also downregulated by nor‐NOHA in hyperoxic group receiving 20 PPM iNO. In conclusion, arginase inhibition may provide a useful therapeutic tool for the increased airway reactivity that occurs in hyperoxia exposed neonates. (IKARIA‐ZAIDS1)
Long‐term endurance training in older adults preserves cardiovagal control but not heart rate responses to exercise
Authors: Luchyshyn Torri A , Norton Katelyn N , Corkal Jamie C , Shoemaker J Kevin
Published: June 2021

Parasympathetic nervous system (PNS) activity decreases with age. Estimates of baroreflex sensitivity (BRS) suggest endurance training may preserve PNS activity in older adults. But, do these estimates reflect the physiologic heart rate responses to exercise? We hypothesized that long term endurance training (ET) in older adults (O) would result in similar levels of PNS control, when compared to young healthy controls (Y). Older ET adults (n = 14; 55 ± 4 years; 4 females) and recreationally active controls (n=12; 26 ± 4 years; 6 females) completed repeated 30‐sec bouts of isometric hand grip exercise (IHG) at 30% of maximal voluntary contraction. Baseline PNS was estimated using cardiovagal BRS (sequence method). The ability to withdraw PNS during IHG was assessed by the reduction in high frequency heart rate variability (HRVhf; wavelet‐based power spectral analysis; ln normalized) and the heart rate (HR) response. Mean BRS (Y: 28.8 ± 18.4ms/mmHg vs O: 23.3 ± 15.2ms/mmHg; P>0.05) and decreases in HRVhf from rest (Y: −0.45 ± 0.63 ms2 vs O: −0.17 ± 0.36 ms2; P>0.05) were not different between groups. However, increases in HR (Y: 7.24 ± 3.36 vs. O: 2.53 ± 1.5 bpm; P=0.0002) from rest were greater in young compared to old. These data suggest that long term ET in older adults can preserve cardiovagal control but not HR responses to a moderate intensity IHG compared to young. Supported by CIHR.
Combined Vasopressin V1a and Oxytocin Receptor Antagonism Prevents Vasopressin‐induced Pressor Response and Sympathoexcitation in Awake Rats
Authors: Rossi Noreen F. , Maliszewska-Scislo Maria
Published: June 2021

Dendritic release of vasopressin (VP) modulates emotional and physiological responses to stress. V1a (V1aR) and oxytocin receptors (OTR) exist in PVN. VP released from dendrites may regulate efferent output from PVN to caudal cardiovascular regulatory sites. We hypothesized that combined inhibition of PVN V1aR and OTR will block mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) responses to PVN VP in awake rats. Sprague Dawley rats were instrumented and studied 2 days later. Injection of 100 ng VP into PVN increased MAP, HR and RSNA (P<0.05). Preinjection with 200 ng V1aR inhibitor elicited a 59% lower pressor response to VP; HR and RSNA decreased below baseline (P<0.05). After 5μg OTR inhibitor, the rise in MAP was 54% lower than with VP alone; HR and RSNA were unchanged. Combined V1aR+OTR inhibition completely blocked the pressor response. The bradycardia and sympathoinhbition seen with V1aR inhibition of VP were prevented. These data suggest that VP activation of PVN V1aR elicits sympathoexcitation and elevates blood pressure. OTR activation by VP within PVN mitigates V1aR responses. Supported by VA Merit Grant.
Vasopressin increases ATF3 mRNA expression in mouse renal inner medulla
Authors: Gao Yang , Cai Qi , Adun Eseoghene , Brooks Heddwen L.
Published: June 2021

In renal inner medulla (IM) the concentration of urea is high and variable depending on the concentrating mechanism. During water restriction (WR), the level of vasopressin increases, followed by further increase of urea concentration in IM. Expression of activating transcription factor 3 (ATF3) is increased by high urea in vitro. To determine whether ATF3 is regulated by high urea in vivo, we water restricted mice for 48 hours and found that ATF3 mRNA increased by 3.81 ± 0.24 fold vs. control 1.00 ± 0.06 (p≤0.05). 7 day infusion of AVP increased ATF3 mRNA by 1.69 ± 019 fold vs. control 1.00 ± 0.15 (p≤0.05). However, 7 day infusion of dDAVP, an agonist of type 2 vasopressin receptor, did not cause significant change in the expression of ATF3 mRNA. ATF3 is an important component in the pathway of endoplasmic reticulum (ER) stress. We previously found that GCN2, a kinase in ER stress pathway, was involved in high urea associated increase of ATF3 in vitro. However, there was no difference in the basal level of ATF3 mRNA between GCN2 null and wild type mice. As in wild type mice, 48 hours of WR increased ATF3 mRNA in IM of GCN2 null mice by 4.14 ± 0.09 fold vs. control GCN2 null mice 1.00 ± 0.06 (p≤0.05). Knocking down GRP78, an important ER chaperone, in mIMCD3 cells did not affect high urea induced increase of ATF3 protein. We concluded that deficiency of ER stress genes does not affect vasopressin induced increase of ATF3 in renal IM.
Relationship of three‐dimensional architecture of thin limbs of Henle's loops to the renal inner medullary concentrating mechanism
Authors: Westrick Kristen Y. , Dantzler William H. , Pannabecker Thomas L.
Published: June 2021

Clusters of collecting ducts (CDs) form the lateral organizing structural motif for the arrangement of thin limbs of Henle's loops and of vasa recta in the renal inner medulla (IM). Within CD clusters (intracluster region), CDs, ascending vasa recta (AVR), and ascending thin limbs (ATLs), form transverse arrays of compartments called interstitial nodal spaces (INSs). The purpose of this study was to investigate quantitatively the relationship of inner medullary thin limbs of Henle's loops to the intracluster region and INSs. With serial section of the IM, Amira imaging software was used to determine the points AQP1‐null descending thin limbs (DTLs) and ATLs enter and exit INSs by tracking DTLs and ATLs as they descended/ascended through the IM. Their distance to the nearest CD was measured on each section. DTLs and ATLs contacting CDs were assumed to lie within the intracluster region, where they may contribute to the generation of an IM osmotic gradient by fluid and solute mixing within an INS. 40 AQP1‐null DTLs were on average 8.6 μm away from their nearest CDs at the base of the IM, and were on average 1.3 μm from CDs at the terminal portion of their length. 21 ATLs were on average 1.79 μm away from CDs as they began to ascend and were 8.02 μm on average from CDs at the IM base. The points at which DTLs and ATLs enter and exit CD clusters is likely related to NaCl reabsorption that occurs at the prebend and ATL.

NIH DK 083338; NSF IOS‐0952885.
PERIPHERAL MECHANISMS INVOLVED IN THE PRESSOR RESPONSE TO L‐PROLINE INTO THE SUPRAOPTIC NUCLEUS
Authors: Azevedo Silvana Lopes , Busnardo Cristiane , de Aguiar Côrrea Fernando Morgan
Published: June 2021

L‐Proline (L‐Pro) shares a number of properties with recognized neurotransmitters and has been postulated to be involved in the cardiovascular control. Magnocellular cells in the hypothalamic supraoptic nucleus (SON) release hormones, which are implicated in the cardiovascular control. In the present study we characterized the cardiovascular responses evoked by the injection of L‐Pro into the SON as well as the peripheral mechanisms involved in these responses. Guide cannulas were placed in the SON. A catheter was introduced into the femoral artery for mean arterial pressure (MAP) and heart rate (HR) recording and the femoral vein for drug injection. Microinjections of 56 nmol/100nL L‐Pro into the SON of the unanesthetized Wistar rats caused pressor and bradycardiac responses. Systemic pretreatment with pentolinium (n= 5) did not block the pressor response evoked by L‐Pro into the SON (ΔMAP: 29.8 ± 2.5 vs. 44.9 ± 7.5 mmHg; t= 2.73, P > 0.05), but the bradycardiac response was blocked (ΔHR: −46.3 ± 1.5 vs. −6.2 ± 3.8 bpm; t= 9.3, P < 0.05). Systemic pretreatment with dTyr(CH2)5(Me)AVP (n= 5) blocked the cardiovascular responses to L‐Pro into the SON (ΔMAP: 30.9 ± 1.8 vs. 2.1 ± 1.7 mmHg; t= 9.2, P < 0.05; and ΔHR: −63.1 ± 4.5 vs. −8.8 ± 8.3 bpm, t= 5.0, P < 0.05). Results suggest that activation of a SON prolinergic pathway causes cardiovascular responses that are systemically mediated by vasopressin release. FAPESP 2010/11303‐6.
From Bohr‐Haldane Effect on Hb‐O2 Binding to Electrical Regeneration in CHD
Authors: Delgado-Almeida Antonio Rafael
Published: June 2021

AIM

Despite extensive clinical and basic research, the precise mechanism for Hb‐O2 binding remains to be determined. Since we have confirmed that ↑RBC K rapidly reverses angina, ST‐T alterations, and induces electrical regeneration of heart in CHD, we tested the hypothesis that RBC K transport is involved on Hb‐O2 binding in human RBC.

Methods/Subjects

Heparin venous sample from healthy subjects were analyzed before (4 ml) and after in Vitro oxygenation (4 ml +1ml 100% O2). In all pH, PO2/PCO2, HCO3 (Stat Profile pHOx), RBC/plasma electrolytes (Na, K, Atomic Emission Spectrophotometry), Cl (Lytek‐Cl). Statistical using Student's t‐test, analysis of variance, p<0.05 as exponential values.

Results

In Vitro blood oxygenation (O2 Sat 87±3 vs 47±6%) resulted in sharply ↓ RBC Ki (87.6± 2.3 vs 92.0±2.3 mmol/lcell, p<4.5E‐09), ↓ RBC Nai (5.8±0.2 vs 6.3±0.2 mmol/lcell, p<6.5E‐05) along with ↓ plasma K (3.3 ±0.3 vs 3.8±0.2 mmol/l, p<9.7E‐05), ↓ Na (134±4 vs 139±2.2, p<1.3E‐03) and ↓ Cl (91±1.6 vs 98.3±1.2 mmol/l, p<7.9E‐11), uncovering a complex biochemical event in Hb‐O2 binding and Bohr Effect, which excludes a simple gas diffusion.

Conclusion

This observation strongly support our novel therapeutic approach improving RBC K, and probably myocardial O2 transport in CHD. Second, it is proposed that RBC K exchange should be included in evaluation of Bohr‐Haldane Effects in clinical and experimental human Hb‐O2 exchanges.
Crystalloid versus colloid resuscitation in a lethal rat model of trauma/hemorrhagic shock
Authors: Huby Maria , Salsbury John , Baer Lisa , Pawelczyk Nicholas , Matijevic Nena , Wang Yao-Wei , Holcomb John , Wade Charles
Published: June 2021

Hemorrhagic shock due to trauma is a leading cause of death. Controversy of which resuscitative fluid may be best to provide adequate tissue perfusion and prevent post‐resuscitation complications still exists. The purpose was to emphasize the superiority of fresh frozen plasma as a resuscitative fluid which restores acid base equilibrium with less volume and lower mortality. Sprague Dawley rats were subjected to trauma and hemorrhagic shock (MAP of 35mmHg) until decompensation. Resuscitation was done with lactated Ringer's (LR, n=15) or fresh frozen rat plasma (FFP, n=16) to maintain MAP at 80mmHg over 3 hours. Total volume of fluid infused were recorded and compared. Metabolic and hemodynamic parameters and tissue H2O content were measured. The volume of blood removed was not different between groups (10ml) nor was time to initiate resuscitation (LR:53±2min, FFP:58±2min). Fluids were able to restore and achieve the target MAP. Mortality was 47% LR and 6% in FFP. However in the surviving animals the volume of LR necessary to achieve target MAP was 7 times the volume of blood loss, whereas 1.1 times was required for FFP. Buffer capacity of FFP was better than LR based upon correction of pH and base excess. Wet‐to‐dry ratios of lung, skin and muscles were reduced with FFP when compared to LR. LR restored normal hemodynamic conditions; however FFP is more efficient and required less volume. (UT Health Houston‐CETIR Dept. Grant)
Chemical Proteomics‐Based Analysis of Off‐target Binding Profiles for Rosiglitazone and Pioglitazone: Clues for Assessing Potential of Cardiotoxicity
Authors: Hoffmann Brian Robert , El Mansy Mohamed , Sem Daniel , Greene Andrew
Published: June 2021

The insulin‐sensitizing drugs rosiglitazone and pioglitazone are currently the only two thiazolidinedione (TZD)‐based drugs on the market to treat type II diabetes in the U.S. Both drugs are thought to be selective for the peroxisome proliferator‐activated receptor gamma. Despite their chemical and mechanistic similarities, there are large cohort studies suggesting rosiglitazone causes congestive heart failure and peripheral edema to greater extent than pioglitazone. This study was performed to complete a mechanistic comparison between the two drugs. The common functional core between the drugs, glitazone, was used as a privileged scaffold upon which rat heart proteins were bound and eluted with glitazone, rosiglitazone, or pioglitazone. Liquid chromatography tandem mass spectrometry was used to determine the drug binding profiles. Results suggest that both drugs bind specific ion channels and modulators, with implications in congestive heart failure, arrhythmia, and peripheral edema. Additionally, numerous proteins involved in fatty acid metabolism, glycolysis/gluconeogenesis, neuronal synaptic transduction, and energy production were detected. While there were slight differences in off‐target binding profiles between rosiglitazone and pioglitazone, there were more similarities in individual protein and pathway comparisons, suggesting similar mechanisms of action and risk profile.
Leukocyte O‐GlcNAcylation: a novel tool for the early detection of type 2 diabetes mellitus
Authors: Springhorn Clare , Essop M Faadiel
Published: June 2021

Hyperglycemia intensifies flux through the hexosamine biosynthetic pathway (HBP), increasing O‐GlcNAcylation of target proteins (a post‐translational modification). The HBP acts as a “fuel sensor” and regulates O‐GlcNAcylation, subject to intracellular glucose availability. This reversible modification is controlled by two enzymes, O‐GlcNAc transferase (OGT), that attaches O‐GlcNAc to protein residues and O‐GlcNAcase (OGA), responsible for O‐GlcNAc removal. Since O‐GlcNAc protein sites are found in leukocytes, we hypothesized increased OGlcNAcylation of leukocyte proteins in the pre‐diabetic milieu. Participants fitting normal, pre‐diabetic or diabetic criteria were recruited through the Bellville South Study (Tygerberg, South Africa). Leukocytes were isolated and the degree of O‐GlcNAcylation, OGT and OGA protein levels evaluated by flow cytometry, Western blotting and fluorescence microscopy. We found significant increases in O‐GlcNAcylation and OGlcNAc/OGA ratios in pre‐diabetic and diabetic subjects (fasting blood glucose, A1C) vs. normal individuals. The O‐GlcNAc/OGA ratio also showed significant differences between pre‐diabetic and diabetic volunteers, representing a sensitive marker for prediabetes. No significant changes were observed for OGT levels. Our data show great promise for a novel diagnostic tool for the earlier detection and management of type 2 diabetes.
Cardiac Linker Histones Are Differentially Regulated Following Hypertrophic Stimuli
Authors: Parvatiyar Michelle S. , Franklin Sarah , Chen Haodong , Wang Yibin , Vondriska Thomas M.
Published: June 2021

Acute or chronic stress causes adaptations in the mammalian heart that result in hypertrophic growth. This growth program involves re‐expression of genes normally silenced during development through mechanisms that are incompletely understood. To investigate the role of global chromatin remodeling in hypertrophy, we performed proteomic analyses to assess chromatin‐bound proteins in basal and hypertrophied mouse myocardium (aortic banding‐induced pressure overload). Histone H1 variants, also called linker histones, which are responsible for higher order chromatin structure, were found to change in quantity, specifically an increase in H1.0, as the heart transitions to failure. We knocked down individual H1 variants by siRNA in neonatal rat ventricular myocytes (NRVMs) and assessed their response to treatment with hypertrophic agonists (1 μM Isoproterenol (Iso), 2 nM Phenylephrine (PE) and 10 μM Endothelin‐1 (ET‐1)). Hypertrophic stimulation of NRVMs increased cell size and changed content and distribution of individual H1 histone variants. Using RT‐PCR an overall reduction was seen in total H1 transcript levels whereas H1.0 variant levels were increased in PE, but decreased in ET‐1, treated cells. Combined with analysis of fetal gene expression in the same conditions, these data suggest linker histones have isoform‐specific roles in cardiac phenotype and chromatin packing. Supported by NIH/NHLBI
Role of histone deacetylases in regulation of NF‐E2‐related factor 2, kruppel‐like factor 2, and cell cycle in vascular endothelial cells in response to disturbed flow
Authors: Chiu Jeng-Jiann , Lee Ding-Yu , Chien Shu
Published: June 2021

Vascular endothelial cells (ECs) are exposed to different patterns of blood flow (disturbed vs. laminar) and the associated shear stresses (oscillatory [OSS] vs. pulsatile [PSS]), which lead to differential EC responses. We investigated the roles of classes I and II histone deacetylases (HDACs, i.e., HDAC‐1/2/3 and HDAC‐5/7, respectively) in regulating NF‐E2‐related factor‐2 (Nrf2) and Krüppel‐like factor‐2 (KLF2), two important transcription factors that govern many shear‐responsive genes, and cell cycle in ECs in response to OSS. Application of OSS (0.5±4 dynes/cm2) to ECs up‐regulated both classes I and II HDACs and their nuclear accumulations, whereas PSS (12±4 dynes/cm2) induced phosphorylation‐dependent nuclear export of class II HDACs. OSS induced the association of HDAC‐1/2/3 with Nrf2 and HDAC‐3/5/7 with myocyte enhancer factor‐2, and the deacetylations of these HDACs led to down‐regulations of the antioxidant gene NAD(P)H quinone oxidoreductase‐1 (NQO1) and KLF2 in ECs. Transfecting ECs with HDAC‐1/2/3‐ and HDAC‐3/5/7‐specific small interfering RNAs eliminated the OSS‐induced down‐regulations of NQO1 and KLF2, respectively. OSS up‐regulated cyclin A and phospho‐retinoblastoma and down‐regulated p21CIP1 in ECs and hence their proliferation; these effects are mediated by HDAC‐1/2/3. The OSS‐induced HDAC signaling and EC responses are mediated by phosphatidylinositol 3‐kinase/Akt. Immunohistochemical examinations of the experimentally stenosed rat abdominal aorta showed high levels of HDAC‐2/3/5 in the EC layer at post‐stenotic sites, where OSS occurs. Our findings indicate the importance of different HDACs in regulating endothelial oxidative, inflammatory, and proliferative responses to disturbed flow with OSS.
Endothelial cell responses to laminar flow: changes in gene expression, protein, and glycocalyx
Authors: Meledeo Michael Adam , Bynum James A , Sondeen Jill L , Bowman Phillip D
Published: June 2021

Previous studies have shown that endothelial cells (ECs) respond to laminar flow and behave more like ECs in vivo. The purpose of this study was to determine the effects of laminar flow on endothelial cells from large compared to small vessels with gene expression analysis, western blotting, and confocal microscopy. Human umbilical vein endothelial cells and dermal microvascular endothelial were grown in Ibidi μ‐VI chamber slides and exposed to 10 or 0.1 dynes/cm2 or static conditions for 24 h. RNA from replicate chambers were isolated, labeled and hybridized to Agilent human whole genome microarrays. Western blots from the same samples were prepared and probed for Syndican‐2, CD31, CD44, CD31 and heparan sulfate. Fixed specimens were prepared for confocal microscopy. Microarray analysis showed that several hundred genes were either up‐ or down‐regulated when ECs were subjected to laminar flow of 10 dynes/cm2 compared to static or low flow. These included gene networks associated with cell morphology, cellular movement, angiogenesis, and inflammation. Changes in cell morphology and rearrangement of glycocalyx elements were observed following flow treatment in both ECs.

Funding provided by USAMRMC.