bluuebunny/crossref_metadata_2025 huggingface dataset:
Wound repair in type I alveolar epithelial cells involves lysosome secretion followed by initiation of actin repolymerization
Authors: Godin Lindsay Marie , Vergen Jorge , Prakash Y.S. , Hubmayr Rolf
Published: June 2021

Stress failure of alveolar epithelial cells is a common feature of ventilator‐injured lungs. While some injured cells undergo necrosis, others repair their plasma membrane (PM) wounds through non‐secretory exocytosis of intracellular vesicles (e.g. lysosomes) to and cytoskeleton remodeling at and around the wound. We measured the timing of lysosomal trafficking and F‐actin reorganization following micropuncture injury in primary type I alveolar epithelial cells (ATI).

ATI were isolated from female rats and loaded with Calcein AM, N‐rhodamine phosphatidylethanolamine (lysosome membrane label) or transfected with LifeAct (GFP tagged actin label). Changes in fluorescence intensity were indicators of time to wound repair (cessation of calcein fluorescence decay following injury), lysosomal trafficking to the wound site, and actin remodeling.

ATI repaired wounds within 27.9±3.9s. Recruitment of lysosomes to the wound site was detected at 2.9±1.5s following injury and preceded actin polymerization by 23.7±5.4s. Failure to repair PM wounds was associated with a weak or absent actin polymerization response.

We conclude that local actin polymerization is a critical step in PM wound repair and postulate that adhesive interactions between PM lipids and the remodeled actin cytoskeleton serve as a driving force for wound closure by promoting lateral PM lipid bilayer flow.

Support: HL63178
HMGB1 accelerates alveolar epithelial repair via an IL‐1β‐dependent activation of TGF‐β1 by the αvβ6 integrin.
Authors: Roux Jeremie , Koh Hidefumi , Fang Xiaohui , Houseman Benjamin T , Shokat Kevan M , Sheppard Dean , Matthay Michael A , Pittet Jean‐Francois
Published: June 2021

High mobility group box 1 (HMGB1) protein, recently described as a proinflammatory mediator of acute lung injury, is a danger‐signaling molecule shown to be actively secreted or passively released from damaged cells. We hypothesized that HMGB1 would accelerate the re‐epithelialisation of the distal lung epithelium, a mechanism that is crucial for the recovery from acute lung injury. We found in the present study that HMGB1 is released in the extracellular space by alveolar epithelial cells that underwent a scratch wound. HMGB1 increased primary rat and human alveolar type II cell monolayer wound repair via an IL‐1β‐dependent activation of TGF‐β1. Specifically, HMGB1 induced the release of IL‐1β that caused the activation of TGF‐β1 via a p38 MAPK, RhoA and αvβ6 integrin ‐dependent mechanism. Furthermore, active TGF‐β1 accelerated the wound closure of primary rat epithelial cell monolayers via a PI3Kα‐dependent mechanism. In conclusion, this study demonstrates that HMGB1 accelerates wound closure in the distal lung epithelium via the IL‐1β‐induced αvβ6‐dependent activation of TGF‐β1, and could thus play a critical role in the resolution of acute lung injury by promoting repair of the injured alveolar epithelium.
Age‐dependent changes in gene expression profiles of postnatally developing rat lungs exposed to nano‐size and micro‐size CuO particles
Authors: Cormack Meredith , Lin Miao , Fedulov Alexey , Mentzer Steve J , Tsuda Akira
Published: June 2021

We hypothesized that acute exposure of developing lungs to fine & ultrafine particles interferes with normal lung development. To test this hypothesis, we performed the following experiments. F‐344 rats [7, 14, >90 days(adult)] were exposed to intranasal instillations of nano‐, micro‐CuO particle suspensions or vehicle (PBS) alone. 48 hours post exposure we performed bronchoalveolar lavage (BAL) in some rats, and lung RNA extraction in others. BAL analyses showed consistent inflammatory response in 7 days‐old neonates and adult rats in response to both nano‐ and micro‐size CuO particles, but not to vehicle alone (PBS). 14‐days old rats did not respond to either size of CuO particles or PBS. Total RNA from unlavaged lungs were analyzed via rat RT2 Profiler PCR array. Both medkine‐heparin‐binding growth factor and c‐fos induced growth factor (also known as VEGF‐D) are up‐regulated in neonatal rats compared to adults, regardless of particle exposure. The pro‐inflammatory chemokine Cxcl2 is consistently up‐regulated in neonatal rats (7, 14 days‐old) comparing to same age PBS instilled group, when they are exposed to both nano‐ and micro‐CuO particles. In summary, PCR analysis in combination with BAL cell counts revealed age‐dependent lung inflammatory response to CuO particles; these methods can be used further to investigate the effects of particle exposure on the postnatal lung development at the molecular level.
HIV‐1‐related proteins decrease Nrf2 expression and alter tight junction protein localization in the plasma membranes of cultured alveolar epithelial cells
Authors: Fan Xian , Joshi Pratibha , Koval Mike , Guidot David M
Published: June 2021

There is abundant evidence that HIV‐1‐related proteins can directly induce oxidative stress and impair tight junction (TJ) protein expression, causing serious consequences for the blood‐brain endothelial barrier and the alveolar epithelial air‐liquid barrier. Nrf2 is the redox‐sensitive transcription factor that regulates the expression of multiple antioxidants. We hypothesized that HIV‐1‐induced oxidative stress and barrier dysfunction could involve changes in Nrf2 expression. To test this hypothesis, we first determined that the gene and protein expression of Nrf2 and of the TJ proteins, occludin and ZO‐1, were significantly decreased in primary alveolar epithelial cells (AECs) isolated from HIV‐1 transgenic rats when compared to AECs from wild type (WT) littermates as assessed by real‐time PCR and western blot analysis. In parallel, exposing rat lung epithelial L2 cells to HIV‐1‐related proteins in vitro significantly inhibited Nrf2 gene expression. We then employed RNA silencing of Nrf2 in AECs isolated from WT rats to determine how this affected occludin and ZO‐1 expression. Consistent with our hypothesis, transfecting these cells with Nrf2 siRNA decreased the membrane localization of occludin and ZO‐1as assessed by immunocytochemistry, even though gene expression of occludin and ZO‐1 did not appear to be affected. Taken together, these data suggest that HIV‐1‐related proteins inhibit the expression of Nrf2 in the alveolar epithelium, and that as a consequence, tight junction assembly in the plasma membrane is somehow impaired. Whether this effect on TJ is mediated by oxidative stress is as yet unknown, but we speculate that Nrf2 expression is a redox‐sensitive target of HIV‐1‐related proteins that could mediate some of the cellular dysfunction in cells that are not directly infected by HIV‐1.
Impact of untranslated regions (UTRs) on differential translation among SftpA1 and SftpA2 gene variants
Authors: Silveyra Patricia , Wang Guirong , Floros Joanna
Published: June 2021

Surfactant protein A (SP‐A) plays an important role in host defense and surfactant‐related functions. In humans, SP‐A is encoded by two genes (SftpA1, SftpA2). Structural and functional differences between SftpA1 (SP‐A1) and SftpA2 (SP‐A2) products have been observed; as well as splice and sequence variation, respectively, at the 5′UTR and 3′UTR. Here, we studied the role of SP‐A 5′UTR and 3′UTR variants, and polyA on translation. We generated constructs (n=14) with the luciferase reporter gene flanked by SPA 5′UTR (SP‐A1: AD′; SP‐A2: ABD, ABD′) and/or 3′UTR (SP‐A1: 6A2, 6A4; SP‐A2: 1A0, 1A3). mRNA was obtained in vitro and polyA was added or omitted. NCI‐H441 cells were transfected and luciferase activity was measured.

Results

1) in the absence of 3′UTR, the SP‐A 5′UTRs exhibit an additive effect with polyA and stimulate translation with different efficiencies (ABD=ABD′>AD′> no 5′UTR); 2) in the absence of 5′UTR, 3′UTR increased translation; with no differences among variants; 3) the combination of SP‐A 5′UTR and 3′UTR showed an additive effect on translation. In this context, the SP‐A1 6A4 3′UTR variant showed higher translation than 6A2, whereas no differences were observed between SP‐A2 3′UTRs (1A0, 1A3).

Conclusion

The 5′UTR, 3′UTR, and polyA contribute to the differential translation of SP‐A1 and SP‐A2. However, the SP‐A1 3′UTR appears to regulate variant‐specific translation. (Supported: NIH HL‐34788)
Function of the HVCN1 proton channel in airway epithelia and a naturally occurring missense mutation, M91T
Authors: Fischer Horst , Iovannisci David , Hajighasemi Mohammad , Illek Beate
Published: June 2021

Airways secrete considerable amounts of acid. In this study we investigated the identity and the pH‐dependent function of the apical H channel in the airway epithelium. In pH stat recordings in Ussing chambers, Zn‐sensitive acid secretion was activated at a mucosal threshold pH~7, above which it increased pH‐dependently at a rate of 339 ± 34 nmole/h.cm per pH unit. Similarly, H currents measured by patch clamping were readily blocked by Zn and activated by an alkaline bath pH. siRNA‐mediated knock down of HVCN1 expression in JME cells resulted in total loss of H currents in patch clamp recordings. Cloning of the open reading frame of HVCN1 from primary human airway epithelia resulted in a wild type clone and a mutant clone M91T HVCN1. The activation of acid secretion in epithelia that natively expressed M91T HVCN1 required ~0.5 pH units more alkaline mucosal pH values compared to wild type epithelia. Similarly, activation of H currents across recombinantly expressed M91T HVCN1 required significantly larger membrane depolarization and pH gradients compared to wild type HVCN1. This study provides functional and molecular indications that the HVCN1 H channel mediates pH‐regulated acid secretion by the airway epithelium. Funded by CFF and NIH HL86323/HL89196
Carotid body O2 sensitivity during chronic hyperoxia and after recovery in normoxia
Authors: Bavis Ryan W. , Pradhan Nelish , Nawreen Nawshaba , Donnelly David F.
Published: June 2021

Developmental hyperoxia attenuates the hypoxic ventilatory response (HVR), an effect which persists into adulthood despite return to normoxia. Part of this impairment is due to carotid body hypoplasia and loss of afferent neurons, but part may be due to impaired responses of the surviving cells. To investigate chemoreceptor function during hyperoxia and after return to normoxia, rat pups were exposed to 60% O2 from birth through P7 and then returned to normoxia until P14. Single‐unit responses to hypoxia were recorded in vitro at ages P1‐P7 (hyperoxia) and at P10 and P14 (recovery). Hyperoxia reduced chemoreceptor responses during acute hypoxia by 4–5 days hyperoxia treatment, consistent with an earlier study (Donnelly et al., Respir. Physiol. Neurobiol. 68:189–197, 2009). Interestingly, these responses recovered to control levels within one week of return to normoxia. These data indicate that carotid chemoreceptor responses to hypoxia are labile during the postnatal period. Moreover, these data do not support a role for reduced O2 sensitivity of surviving cells in long‐lasting reduction of the HVR after developmental hyperoxia. Supported by NIH grants HL‐083972, RR‐016463 (Maine INBRE) and HL‐073500.
Effects of IV Remifentanil (Remi) on the discharge patterns of canine pre‐Botzinger complex (pBC) neurons
Authors: Mustapic Sanda , Radocaj Tomislav , Stucke Astrid G. , Stuth Eckehard A. , Dogas Zoran , Hopp Francis A. , Zuperku Edward J.
Published: June 2021

Mu‐opioids at clinical doses typically produces bradypnea, which may be mediated by their effects on presynaptic neurons that supply inputs to pBC neurons. This study evaluated the effects of the rapid, short‐acting mu‐opioid Remi (IV) on the discharge patterns of pBC neuron subtypes in decerebrate, vagotomized, mechanically‐ventilated, paralyzed dogs during hyperoxia. A NeuroNexus 16‐electrode microprobe was used to simultaneously record from multiple pBC neurons before, during and after the IV infusion of Remi. Histograms (CTHs) triggered from the phrenic neurogram (PNG) were used to determine peak and time‐averaged discharge frequency (Fn) and profiles. Remi (0.94 mcg/kg/min) increased TI and TE by 3% and 71% respectively, and decreased peak PNG by 55%. Depression (peak Fn) among subtypes was not different: I‐aug (28±10%; n=32), I‐dec (22±10%; n=18), E‐aug (18±10%; n=15), and E‐dec (38±8%; n=26). Depression based on average Fn was similar. The magnitude of neuronal depression was not related to TI and TE or control Fn. Differential depression between simultaneously recorded neurons was common. While the pBC neurons were much less depressed than the peak PNG, the similar degree of depression of neuron subtypes and response variability did not allow bradypnea to be assigned to any pBC neuron subtype. The mechanism for bradypnea may lie outside the pBC. Supported by VA Med. Res. and NIH GM059234 funds.
Cardiovascular responses to standing and walking in earth and simulated moon and mars gravities
Authors: Evans Joyce M , Kilfoil Peter J , Wang Siqi , Mohney Lindsay , Knapp Charles F
Published: June 2021

Introduction

Human cardiovascular responses to standing and walking in simulated reduced gravity were measured using a treadmill enclosed in a lower‐body positive pressure (LBPP) chamber (AlterG device).

Methods

Heart rate, HR, continuous blood pressure, BP, (Portapres), segmental fluid shift (THRIM), diastolic function and stroke volume (Biosounds) were recorded on 7 subjects (4 males, 3 females) at supine and while standing and walking under 100% (earth), 40% (mars), and 20% (moon) body weights (BW).

Results

Subjects standing at 20% and 40% BW had reduced HR (p<0.01), increased systolic BP (p<0.05) and increased thoracic impedance (p<0.001) compared to standing. Reduced BW was associated with increased upper and lower leg impedance (loss of fluid volume, p<0.05). Stroke volume was greatest while supine, at 20%, 40% BW and walking compared to standing (p<0.01). Mitral E to A ratio was reduced for 20%, 40%, and 100% BW compared to supine (p<0.01). The number of blood pressure ramps and baroreflex sequences decreased as body weight was reduced.

Conclusion

Fluid redistribution in the body and short‐term cardiovascular changes induced by Alter‐G unloading are compatible with those resulting from exposure to microgravity. Supported by KY NASA EPSCoR 52611.
Reconsideration of exercise prescription as the countermeasure for prevention of muscle atrophy in space
Authors: Ohira Takashi , Okabe Hirooki , Kawano Fuminori , Oke Yoshihiko , Fujita Ryo , Nomura Sachiko , Nakai Naoya , Ohira Tomotaka , Ohira Kazutaka , Ohira Yoshinobu
Published: June 2021

Responses of electromyogram (EMG) activities in soleus and vastus lateralis (VL) to various types of exercise were examined in human subjects. The soleus EMG activity during walking was gradually increased in subject whose heel lands on the treadmill first, when the speed and uphill inclination were increased. But in some subjects whose front sole lands first, soleus was more active when they were walking slowly on a flat treadmill. Greater EMG activities of soleus, but not VL, were observed during pedaling or lifting weight on the floor by using their front sole, vs. arch or whole sole, in all subjects. Utilization of VL, in which EMG level was greater during cycling than walking, was stimulated, when the load of bicycle ergometer was increased. It was suggested that muscle specific exercise should be performed as the countermeasure. This study was supported, in part, by Grant‐in‐Aid for Scientific Research S (19100009, YO) from Japan Society for the Promotion of Science.
A questionable association of stroke volume and arterial pulse pressure under gravitational stress
Authors: Cote Anita T , Bredin Shannon S.D. , Warburton Darren E.R.
Published: June 2021

Arterial pulse pressure (PP) has been shown to be correlated strongly with stroke volume (SV) when humans are subjected to a gravitational stress. The purpose of this investigation was to assess the PP and SV response to progressive lower body negative pressure (LBNP) using individual responses rather than the mean response per stage of LBNP as reported previously. Sixteen healthy men and women (Age(SD) = 24.4(3.0)) were assessed during a 12 min baseline collection, during four 12‐minute stages of progressive LBNP at −15, −30, −45 and −60 mm Hg, and during 12 min of recovery. Hemodynamic measures were compared from baseline throughout −60 mm Hg. Systolic blood pressure (105 ± 7.8 to 103± 8.3 mm Hg) and mean arterial pressure were not statistically different (81 ± 5.6 to 83 ± 5.9 mm Hg); however, there was a significant difference for diastolic blood pressure (69 ± 5.3 to 72 ± 5.8 mm Hg), PP (37 ± 5.7 to 31 ± 7.0), SV (80 ± 17.0 to 26.6 ± 10.0) and heart rate (63 ± 8.3 to 86 ± 14.2). Typical group analysis revealed a strong positive relationship between LBNP and SV (r2= 0.99), LBNP and PP (r2= 0.96), and SV and PP (r2= 0.97). When comparing the individual slopes there was a more modest relationship (r2 = 0.53). Our results show high individual variability in response to LBNP and the need to consider individual PP and SV responsiveness. Natural Sciences and Research Council of Canada, Canadian Institutes of Health Research, Michael Smith Foundation
The effect of simulated 1/6th and 1/3rd gravity on gastrocnemius muscle mass and fractional protein synthesis rates in mice
Authors: Wiggs Michael Parker , Swift Joshua M , Lima Florence , Greene Elizabeth S , Bloomfield Susan A , Fluckey James D
Published: June 2021

During periods of disuse or unloading (0G), there is a substantial decline in mass and function of skeletal muscle. However the effects of partial weight bearing, similar to Lunar (G/6) or Martian (G/3) gravitational fields, have yet to be determined. We used a partial G mouse model to assess graded reductions in weight bearing on skeletal muscle. We hypothesized that mice exposed to 21 days of G/6 and G/3 would experience significant reductions of gastrocnemius (gastroc) mass compared ambulatory control animals (1G), but that the magnitude of these changes would be less than in 0G mice. Further, decrements of fractional protein synthesis rates (FSR) would be consistent with reductions of muscle mass. Female BALB/cByJ (4 month old; n=43) mice were randomly assigned to fill four groups (1G, 0G, G/6 or G/3). After a 21‐day suspension period gastroc muscles were excised and weighed, and cumulative FSR was assessed using deuterium oxide. Total body mass was similar among groups (p>0.05). However, wet weight of gastroc was lower in G/6 and G/3 compared to 1G (p<0.05), and even lower in HU (p<0.05 vs 1G, G/6 and G/3). FSR was not different among 1G, G/6 and G/3, but was lower in 0G animals (p <0.05). This study shows that muscle mass is related to applied load but partial loading is able to maintain FSR.

Funded by National Space Biomedical Research Institute via NASA Cooperative Agreement NCC 9‐58
Exercise training improves the metabolic phenotype of pre and postnatal growth restricted male rat offspring
Authors: Laker Rhianna C , Wlodek Mary E , Wadley Glenn D , McConell Glenn K
Published: June 2021

Fetal growth restriction impairs glucose tolerance. We determined whether exercise training early or later in life normalizes the metabolic phenotype in adult male rats born small or postnatally growth restricted. Growth restriction induced on day 18 of pregnancy yielded growth Restricted (Restrict 10–15% smaller) compared to sham‐operated Control (Con) and sham‐operated Reduced litter (Red) pups. Male offspring were sedentary or underwent early (5–9 wks of age) or late (20–24 wks) exercise training (treadmill running 5 day/wk 60 min/day). A fasted ip glucose tolerance test (GTT 1g/kg) was performed 48 hrs after the last exercise bout at 9 or 24 wks. At 9 wks HOMA‐IR was not different between sedentary groups and was lower after early exercise in all groups, suggesting improved insulin sensitivity. At 24 wks Restrict and Red had higher HOMA‐IR compared to Con. First phase insulin secretion tended to be lowest in sedentary Restrict (75–85%) and increased with late exercise training in Restrict (80%). Despite normal glucose, late exercise Con had lower insulin during GTT compared to Con sedentary and early exercise while early exercise Red had lowered insulin compared to sedentary Red at 24wks. In summary early exercise improved adult insulin sensitivity in postnatal growth restricted rats. The 85% reduction in insulin secretion, associated with β‐cell mass deficits was restored with late exercise in males born small.
Treatment with swimming and treadmill exercises reduced neuropathic pain on chronic constriction injury of sciatic nerve of rats
Authors: Li Yung Tsung , Li Zong Ying , Hsueh Meng I , Hung Hui Chun , Ou Hsiu Chung , Chen Yu Wen
Published: June 2021

BACKGROUND

Treatment of neuropathic pain is still a challenge. Pharmacotherapies provide an effective efficacy for treatment of neuropathic pain, but drugs produce some side effects. Whether exercise training without drug treatment could provide beneficial effects on neuropathic pain.

MATERIALS AND METHODS

Chronic constriction injury (CCI) of the sciatic nerve in male SD rats produces behavioral hypersensitivity and is an accepted model of neuropathic pain. All rats were divided into 6 groups: sham, sham with swimming/treadmill exercise, CCI, CCI with swimming/treadmill exercise. We compared behavior responses and body weight in sham operated and CCI rats with or without exercise training. Thermal hyperalgesia and tactile allodynia was assessed by Plantar test and von Frey filament test.

RESULTS

Our results showed that treatment with swimming and treadmill exercises attenuated thermal hyperalgesia and mechanical allodynia significantly in the CCI rat. The change trend of body weight indicated no significant differences between each group but not the sham group.

CONCLUSION

Our preliminary results support both swimming and treadmill exercises training attenuated neuropathic pain in the rat. This study was supported partially by a grant from the China Medical University (CMU98‐S‐35) of Taiwan.
Exercise Training in Two Kidney One Clip Rats Decreases Blood Pressure via Predominantly Nitric Oxide Mechanisms in Paraventricular Nucleus
Authors: Rossi Noreen F. , Maliszewska‐Scislo Maria
Published: June 2021

Exercise training (ExT) decreases MAP in hypertension but the mechanism in the two kidney‐one clip (2K1C) model with Ang II and sympathetic components is not known. Plasma Ang II acts on circumventricular organs that project to paraventricular nucleus (PVN) whose efferent output influences sympathetic tone. We hypothesized that voluntary wheel ExT in 2K1C rats for 6 or 12 wk would enhance GABA and nitric oxide (NO) sympathoinhibitory mechanisms in the PVN. Bicuculline (BIC 200 pmol) or L‐NAME 200 μg was microinjected into PVN in awake sedentary (SED) or ExT sham clipped (SC) and 2K1C rats. At 6 wk ExT in SC or 2K1C did not change resting MAP or HR vs SED groups. BIC evoked greater rise in MAP and HR in SC ExT vs SED but not in 2K1C rats. After BIC, RSNA increased similarly in SED and ExT in both SC and 2K1C. At 12 wk resting MAP was 27 mmHg lower in 2K1C ExT vs SED rats (P< 0.05). After L‐NAME in 2K1C, RSNA increased 24±9% in SED vs 79±16% in ExT (P<0.02). After BIC, RSNA increased less in 2K1C ExT vs SED rats (P<0.01). No differences in responses to L‐NAME or BIC occurred in SC SED vs ExT rats at 12 wk. In 2K1C rats when hypertension is not Ang II dependent (12 wk), ExT significantly decreases MAP. 12 wk but not 6 wk ExT in 2K1C rats exerts an inhibitory effect on RSNA mediated at least partly by PVN nitritergic mechanisms. After longer ExT inhibitory actions of NO on excitatory neurotransmitters/pathways in PVN may predominate over those for GABA. HL‐079102
Early exercise training attenuates left ventricular dysfunction, myocardial infarction area and molecular abnormalities in diabetic rats
Authors: Rodrigues Bruno , Jorge Luciana , Malfitano Christiane , Medeiros Alessandra , Rosa Kaleizu Teodoro , Alba‐Loureiro Tatiana , Candido Georgia Orsi , Curi Rui , Lacchini Silvia , Brum Patricia Chakur , De Angelis Kátia , Irigoyen Maria Cláudia
Published: June 2021

In this study we evaluated the effects of early exercise training (ET) on mortality rate, myocardial infarction (MI) area and VEGF gene levels, as well as on left ventricular (LV) function and calcium handling abnormalities in diabetic rats after MI. Male Wistar rats were divided in sedentary (S) and exercise training (ET) groups: control (C), MI+S (SI), MI+ET (TI), diabetes+MI+S (SDI) and diabetes+MI+ET (TDI). After 20 weeks of ET, MI size although similar at the beginning (~40±3%), was reduced at the end of the protocol in trained groups. MI size reduction was associated with increased mRNA VEGF levels in trained animals. Trained rats displayed higher cardiac output and ejection fraction (TI: 59±4 and TDI: 58±4% vs SI: 40±1 and SDI: 54±1%) when compared to sedentary ones. This improvement on LV function in trained animals was associated with increased expressions of SERCA2 (25%) and phospholamban phosphorilated at serine 16 (34%). In addition, survival rate was improved after ET period (TI: 87 vs SI: 41% and TDI: 75 vs SDI: 30%). These results showed that early ET intervention induced MI area reduction, LV function and survival improvement in trained animals. Moreover, the improvement of mRNA VEGF levels and Ca2+ handling proteins gives support to consider ET as a coadjuvant therapy able to induce additional changes at myocardium molecular levels in diabetic subjects after an ischemic event.

CNPq
Endurance exercise protects cardiac tissue from doxorubicin‐induced proteolysis and apoptosis
Authors: Kavazis Andreas N , Smuder Ashley J , Min Kisuk , Tümer Nihal , Powers Scott K
Published: June 2021

Doxorubicin (DOX) is an antitumor agent used in cancer treatment. Unfortunately, the clinical use of this highly effective drug is limited due to cardiotoxicity. We hypothesized that endurance exercise would result in upregulation of cardiac antioxidant enzymes and heat shock protein 72 which would protect against DOX‐induced proteolysis and apoptosis. Adult male rats were divided into sedentary or exercise groups followed by placebo or DOX treatment. Our results indicate that DOX promotes disturbances in cardiac muscle redox balance (i.e. increased levels of 4‐hydroxynonenal and protein carbonyls). Furthermore, the activities of calpain and caspase‐3 were significantly increased in cardiac tissue of sedentary animals treated with DOX. Importantly, exercise prior to DOX treatment blunted the increased cardiac protein oxidation and protease activation. Furthermore, exercise reduced the number of apoptotic nuclei in the heart following DOX treatment. These results suggest that DOX‐induced cardiotoxicity may be attributed to disturbances in cardiac muscle redox balance, increased proteolytic activity, and apoptosis. Importantly, data from these experiments suggest that endurance exercise may act to preserve these detrimental side effects of DOX treatment in cardiac tissue.

Supported by AHA (to ANK), NIH R01 HL087839 (to SKP) and Medical Research Service, Dept. of Veterans Affairs (to NT)
Exercise training restores contractility in collateral‐dependent myocardium independent of calcium sensitization of myofilament proteins
Authors: Sarin Vandana , Wu Xin , Muthuchamy Mariappan , Heaps Cristine L.
Published: June 2021

Cardiac dysfunction is frequently observed in patients and animal models of coronary artery disease. For this study, we hypothesized that exercise training would improve force generation in myocardium distal to chronic coronary artery occlusion via calcium sensitization of myofilament proteins. Ameriod constrictors were surgically placed around the proximal left circumflex coronary (LCX) artery of adult female Yucatan pigs. Twenty‐two weeks post‐surgery myocardium was isolated from control (left anterior descending artery‐dependent) and collateral‐dependent (LCX‐dependent) regions of sedentary (Sed; pen‐confined) and exercise‐trained (Ex; treadmill; 5 days/wk for 14 wks) pigs. Force measurements in myocardial strips showed that percent change in force generated at stimulation frequency of 4 Hz vs. 1 Hz was higher in the collateral‐dependent region of Ex vs. Sed. Time to half peak tension and time to half relaxation in a muscle twitch at 1 Hz was similar in control and collateral‐dependent regions of the Sed and Ex pigs. Calcium sensitivity of cardiac myofilaments was unchanged in control and collateral‐dependent regions of the Sed and Ex pigs as determined by isometric tension‐pCa relationships. Thus, exercise training accentuates force generation in collateral‐dependent myocardium of Ex pigs independently of calcium sensitization of myofilament proteins. Supported by NIH HL064931.
Regulation of blood vessel patterning and guidance
Authors: Eichmann Anne
Published: June 2021

The formation of the vascular system during vertebrate embryonic development is crucial to ensure oxygen and nutrient delivery to all tissues in the organism. Blood vessels are formed by differentiation of primitive mesodermal cells into endothelial cells, which coalesce into primitive tubes that proliferate and migrate to colonize tissues. With the onset of the heart beat and blood flow, primitive vessels are rapidly remodelled into branched networks with a characteristic and reproducible anatomy. The major axial vessels such as the aorta and cardinal veins are common to developing mouse, chick and zebrafish embryos. Formation of a stereotyped architecture ensures efficient oxygen and nutriment delivery and thus function of the vascular system. In addition to growth factor gradients, hemodynamic forces are important in the regulation of pattern formation and cooperate to shape the stereotyped vessel anatomy of vertebrate embryos. In addition, blood vessels are often aligned with nerves and display similar branching patterns in peripheral tissues. The molecular mechanisms regulating common wiring of nerves and blood vessels have attracted considerable interest over the past few years. Developing axons navigate through the embryo by responding to a number of different signals in their immediate environment. Molecules such as Semaphorins, Slits and Netrins and their specific receptors provide key ligand–receptor interactions for this process during both neuronal and vascular development.
Annexin A1 mediates the effects of dexamethasone on macula densa cyclooxygenase‐2 expression
Authors: Seidel Saskia , Roeschel Tom , Kahl Thomas , Schnermann Jurgen , Bachmann Sebastian , Paliege Alexander
Published: June 2021

In the present study we aimed to elucidate the role of the anti‐inflammatory protein annexin A1 (ANXA1) in the regulation of macula densa (MD) cyclooxygenase‐2 (COX‐2) expression by dexamethasone (Dex). To this end, cultured mouse macula densa cells (MMDD1) were treated for 8h with 0.1μM Dex. The presence of ANXA1, COX‐2, the ANXA1 receptor FPRL1, and the ANXA1 transporter ABCA1 was verified by PCR. Dex‐dependent changes in MMDD1 COX‐2 and ANXA1 expression were studied by TaqMan real time PCR. ANXA1 secretion was quantified by Western blotting of cell‐culture supernatant. To study the effects of ANXA1 receptor signalling on macula densa COX‐2 expression cells were treated either with the ANXA1 receptor agonist fMLP (1μM) or the ANXA1 receptor antagonist cyclosporin H (CyH; 0.1μM) for 8h. MMDD1 cells express COX‐2, ANXA1, ABCA1, and FPRL1. Treatment with Dex reduced COX‐2 mRNA abundance to 63±5% of controls (p<.05) whereas ANXA1 mRNA expression and protein secretion were increased to 156±10% and 360±19% of control, respectively (p<.05). COX‐2 mRNA expression was reduced by fMLP (66±6% of control; p<.05) and increased by CyH (183±33% of control; p<.05). In summary we have shown that Dex treatment increases MD ANXA1 expression and secretion. Stimulation of the ANXA1 receptor FPRL1 reduces COX‐2 mRNA expression. Our results thus suggest that the inhibitory effects of Dex on MD COX‐2 expression are mediated by ANXA1.
Structure‐Function Correlation of G6, a Novel Small Molecule Inhibitor of Jak2: Indispensability of the Stilbenoid Core
Authors: Majumder Anurima , Govindasamy Lakshmanan , Magis Andrew , Kiss Robert , Polgar Timea , Allan Robert W. , Agbandje‐McKenna Mavis , Keseru György M. , Bisht Kirpal , Sayeski Peter P.
Published: June 2021

Somatic mutations in the Jak2 allele, such as V617F, cause aberrant Jak‐STAT signalling and can lead to the development of myeloproliferative neoplasms (MPNs). This has led to the search for small molecule inhibitors that target Jak2. Using structure‐based virtual screening, our lab recently identified a novel small molecule inhibitor of Jak2 named G6. Here, we identified a structure‐function correlation of this compound. Specifically, five derivative compounds of G6 having structural similarity to the original lead compound were analyzed for their ability to i) inhibit Jak2‐V617F mediated cell growth ii) inhibit the levels of phospho‐Jak2, phospho‐STAT3 and phospho‐STAT5 iii) induce apoptosis in HEL cells and iv) suppress pathologic cell growth of Jak2‐V617F expressing human bone marrow cells, ex vivo. Additionally, we examined the interactions of these compounds with the ATP binding pocket of the Jak2 kinase domain. We found that the stilbenoid core containing derivatives of G6 significantly inhibited Jak2‐V617F mediated cell proliferation in a time‐ and dose‐dependent manner. They also inhibited phosphorylation of Jak2, STAT3 and STAT5 proteins within cells. Moreover, we report that these stilbenoid derivatives of G6 induced apoptosis and inhibited the pathologic growth of Jak2‐V617F expressing human bone marrow cells, ex vivo. Collectively, our data demonstrate that G6 has a core stilbene structural element which is indispensable for maintaining its Jak2 inhibitory potential. Supported by R01‐HL67277.
Selective downregulation of human Kv1.3 channels by PKA I
Authors: Kuras Zerrin , Kucher Vladimir , Neumeier Lisa , Dillehay Kelsey , Conforti Laura
Published: June 2021

The voltage‐gated K channel Kv1.3 plays an important role in the earliest stages of T cell activation and its inhibition suppresses T cell function. Protein kinase A (PKA) modulates Kv1.3 activity and downregulates T cell function. Two types of PKA are expressed in human T cells, Type I and Type II. Molecular and electrophysiological studies were undertaken to determine the PKA isotype modulating Kv1.3. 8Br‐cAMP (8Br), a non‐selective activator of PKA, inhibits Kv1.3 currents by 35% both in primary human T and in Jurkat cells. This inhibition was prevented by the PKA blocker PKI. Jurkat cells were transfected with siRNAs against the regulatory subunit of PKAI and a GFP plasmid (10:1 ratio) for visual recognition during patch‐clamp experiments. Selective downregulation of PKAI knocks down PKAI but not PKAII by 45% as determined by semiquantitative RT‐PCR. 8Br inhibits Kv1.3 current in scramble siRNA transfected (control) cells by 33%. The response to 8Br was abolished in siRNA‐transfected cells. Further experiments in primary T cells showed that the PKAI selective agonist pair 8‐piperidinoadenosine‐cAMP/8‐hexylaminoadenosine‐cAMP induced Kv1.3 inhibition comparable to 8Br, while the PKAI antagonist Rp‐8‐Br‐cAMP significantly reduced 8Br inhibition. These results indicate that PKAI, and not PKAII, is responsible for the regulation of Kv1.3 channels in human T lymphocytes. (NIH AI083076 and AHA 0855457D)
Increases in N‐type calcium current augment transmitter release following three days of chronic intermittent hypoxia
Authors: Kunze Diana Lee , Ramirez‐Navarro Angelina , Kline David D
Published: June 2021

We previously reported changes in synaptic transmission between chemosensory afferent fibers and secondary neurons in NTS in response to chronic intermittent hypoxia (CIH). Ten days (10D) of CIH in rats produces an increase in the frequency of spontaneous miniature release of transmitter (mEPSC) while the evoked EPSC (eEPSC) is significantly reduced (Kline et al., J. Neurosci. 27:4663, 2007). We attributed the latter to a homeostatic response to the increased mEPSC and postulated that the two responses could be separated in time. Here we compared the eEPSC amplitude in rats subjected to a shorter hypoxic exposure, 3 days CIH with NORM and also compared mEPSC frequency in both groups. After 3D CIH, eEPSC amplitude was slightly elevated, 202±26 vs 182±4 pA. To the contrary, mEPSC release had already increased, 11± 3 (CIH) versus 5±1Hz (NORM) similar to values at 10D CIH (11.7±2.6 Hz). To address mechanisms, in voltage clamp studies we recorded calcium currents from soma of the presynaptic neurons. We compared the amplitude of N‐type calcium current (ω‐conotoxin GVIA‐sensitive) responsible for evoked release in cells isolated from 3D CIH or NORM rats. The current was increased (p<.02) after CIH (65±6 pA/pF) compared to control (42±7 pA/pF) suggesting that an increased functional expression of the N‐type channel may be responsible for the increase in spontaneous release as well as the small increase in evoked release at 3D CIH. HL090886
Decrease in cardiovascular stress reactivity in AT1A receptor knockout mice is stimulus intensity ‐ dependent
Authors: Mayorov Dmitry , Chavez Carolina A , Choy Kwok H
Published: June 2021

Angiotensin AT1A receptor knockout (AT1A−/−) mice have attenuated blood pressure (BP) responses to intense aversive stimuli including restraint or footshock. Conversely, BP responses to less intense aversive events, such as novelty or handling, appear to be unaltered in AT1A−/− mice. In the present study, we examined whether this complexity relates to the intensity of aversive stimulation. AT1A−/−(n=7) and AT1A+/+mice (n=7) were implanted with BP telemetry devices and subjected to 5‐min shaker stress of different intensities (40, 80 and 160 rpm). Shaker stress increased BP in an intensity‐dependent manner in AT1A+/+mice (+21±3, +25±3 and +33±2 mmHg, respectively). The BP rises during the low‐ and mid‐ intensity shaker were not altered in AT1A−/− mice, whereas that to high‐intensity shaker was reduced by 25%. This reduction could not be ascribed to attenuated vascular reactivity to intense sympathetic stimulation, because intraperitoneal administration of the α1‐adrenoreceptor agonist phenylephrine (0.1–5 μg/g) dose‐dependently increased BP in both strains, and these increases were similar at all doses tested. These data suggest that AT1A receptors are essential for the full expression of the BP response to high‐ but not low‐aversive stimuli in mice. This selectivity appears to relate primarily to alterations in central stress processing rather than sympathetic vascular reactivity. NHMRC (Australia)
Age‐dependent failure of cardiac auto‐resuscitation in neonatal Pet‐1−/− mice exposed to multiple bouts of anoxia: implications for the Sudden Infant Death Syndrome
Authors: Cummings Kevin James , Li Aihua , Nattie Eugene
Published: June 2021

Medullary serotonin (5‐HT) defects are strongly associated with SIDS, a disease often culminating in failed cardiac auto‐resuscitation. Pet‐1−/− mice have an altered gasping pattern during a single bout of anoxia (Erickon & Sposato, 2009). We hypothesized that cardiac auto‐resuscitation would be compromised in Pet‐1−/− mice exposed to multiple bouts of anoxia. Using a pneumotach/mask with ECG, we exposed Pet‐1−/− mice and littermates to 4 anoxic bouts (97% N2/3% CO2), separated by 5 min of room‐air at P4–5 (Pet‐1−/−: n=10; littermates: n=12), P7–8 (n=7, 9) and P11–12 (n=8, 11). Anoxia was maintained until hypoxic apnea. In addition to gasp latency, we measured heart rate (HR) at primary apnea and 1st gasp, and the time for 90% HR recovery. There were no between‐genotype differences at P4–5. By P7–8, HR at apnea and 1st gasp was significantly lower in Pet‐1−/− animals (‐25 and ‐32 beats/min, respectively; P<0.05), along with a prolonged gasp latency (P<0.001). Further, HR recovery time was delayed 4‐fold relative to littermates (P=0.01), and in 3/7 Pet‐1−/− animals, HR failed to recover after the 3rd anoxic bout. No defects were observed at P11–12. These data suggest that defects in the medullary 5‐HT system may be a causal factor in SIDS via failure of cardiac auto‐resuscitation. This work was supported by NIH grant HL28066 and HD36379. Support for KJC provided by the Parker B. Francis Foundation.
Responses of nucleus tractus solitarius neurons to vanilloid, purinergic and CCK receptor activation
Authors: Browning Kirsteen N , Travagli R. Alberto
Published: June 2021

Recent reports indicate that cardiovascular sensory inputs to neurons in the nucleus tractus solitarius (NTS) can be distinguished based on their response to vanilloid‐ or to purinergic‐ agonists, active only on C‐ or A‐δ fibers respectively.

The NTS subnucleus centralis (cNTS) receives vagal afferent inputs from subdiaphragmatic viscera and is involved in vago‐vagal circuits controlling gastrointestinal (GI) motility. We have shown that CCK‐8s increases glutamate release from vagal afferent fibers onto cNTS neurons. Since CCK‐8s is thought to exert its GI‐related effects via activation of C‐fibers only, we aimed to test whether vanilloid and CCK‐8s sensitive inputs to cNTS neurons were distinguishable from those responsive to purinergic agonists. Spontaneous EPSCs were recorded from cNTS neurons in response to perfusion with capsaicin (CAP), α‐β‐met‐ATP (ATP) and/or CCK‐8s.

ATP, CAP or CCK‐8s increased sEPSC frequency in 37, 75 and 43% of cNTS neurons, respectively. Approximately 30% of cNTS neurons were responsive to both CCK‐8s and ATP, to CCK‐8s and CAP, or to ATP and CAP while 32% of the neurons were responsive to all three agonists. Interestingly, all neurons responding to ATP or CCK‐8s were also responsive to CAP.

Thus, unlike cardiovascular sensory inputs, C‐ and A‐δ inputs to cNTS neurons do not appear to have selective responses to either ATP or CAP.

Supported by DK 55530
Muscle Metaboreflex‐Induced Coronary Vasoconstriction Limits Left Ventricular Performance‐Blood Flow Relationship
Authors: Coutsos Matthew , Sala‐Mercado Javier Agustine , Ishinose Masashi , Li ZhenHua , O'Leary Donal S
Published: June 2021

Raising myocardial work elicits coronary vasodilation. However, during dynamic exercise with muscle metaboreflex activation (MMA), a rise in sympathetic activity increases cardiac performance as well as induces alpha‐1 adrenergic mediated coronary vasoconstriction. Thus, the increase in coronary blood flow (CBF) is restrained by limited vasodilation. We tested whether this limited increase in CBF during MMA acts to functionally limit increases in left ventricular (LV) performance. Using chronically instrumented dogs we assessed the relationship between left ventricular function and blood flow during exercise and MMA before and after alpha‐1 adrenergic blockade (prazosin 50–100μg/kg). Coronary vascular conductance (CVC) was plotted as a function of cardiac power. Preload recruitable stroke work (PRSW) was assessed vs. CBF. We found that after alpha‐1 blockade the relationship between CVC and cardiac power was shifted upwards with an increase in slope such that MMA elicited greater increases in CVC per increase in cardiac power. Furthermore, PRSW increased linearly with increases in CBF independent of alpha blockade. We conclude that during MMA sympathetic vasoconstriction limits increases in CBF which restrains increases in cardiac contractility and the ability to increase cardiac power. NIH HL‐55473 and HL‐095819.
Age‐related changes in autonomic control during sleep: symbolic and complexity analyses of heart rate variability
Authors: Tobaldini Eleonora , Viola Antoine , Chellappa Sara , Porta Alberto , Casali Karina R , Montano Nicola
Published: June 2021

Scanty data are available on the differences in neural control of circulation during sleep between young (Y) and older (O) subjects. The aim of our study was to assess the age‐related changes of heart rate variability during sleep using symbolic analysis (SA) and entropy indexes, Shannon entropy (SE) and Conditional Entropy (CE). A polysomnography was performed in 12 Y and 12 O healthy volunteers; time series of RR intervals were constructed for each sleep stage (Wake, W; slow wave sleep, SWS; and REM) and analyzed using SA, SE and CE.

SA is based on the conversion of time series into symbols, the construction of patterns and the identification of four families: 0V (no variation), marker of sympathetic modulation, 1V, 2LV and 2UV (two unlike variations), marker of parasympathetic modulation. SE and CE measure the amount of information carried by the new sample when the previous are known. A decrease of entropy indexes is marker of aging and disease.

In the O, REM was characterized by a decrease of SE, CE and 2UV% and an increase of 0V%, with respect to SWS and W. Compared to Y, the O showed in all stages a reduction of SE, CE and 2UV% and an increase of 0V%, and these differences were more evident during REM sleep.

In conclusion, REM is characterized by a decrease of complexity and a predominance of sympathetic modulation in older subjects and it may represent a state of increased risk for cardiovascular events during aging.
AT1A receptor deficiency attenuates cardiovascular reactivity to contextual fear conditioning but not anxiety‐like behavior in mice
Authors: Mayorov Dmitry , Choy Kwok H , Chavez Carolina A
Published: June 2021

Angiotensin AT1 receptors are important in regulating blood pressure (BP) reactivity to unconditioned aversive stimuli. In this study, we examined the influence of AT1A receptors on cardiovascular and behavioral effects of contextual fear conditioning. AT1A receptor knockout (AT1A−/−) and AT1A+/+ mice were implanted with BP telemetry devices and subjected to a 5‐min footshock session consisted of 4 brief electric footshocks. Mice were then re‐exposed to the same context (the footshock chamber) 4 hours and 1–4 days after the footshock, and their behavior was analyzed by a computerized video‐tracking system. The BP rise during the re‐exposure sessions was consistently lower in AT1A−/− than AT1A+/+mice (+24±4 and +37±2 mmHg, respectively, n=5–6). However, immobility duration (freezing) was similar between groups. Unexpectedly, AT1A−/− mice displayed increased anxiety‐like behavior as evidenced by reduced time spent in the centre of the footshock chamber. Likewise, the BP rise during the elevated plus maze test was lower by 25% in AT1A−/− mice, although anxiety scores increased. These data indicate that AT1A receptor deficiency in mice attenuates the BP responsiveness to conditioned contextual fear. This attenuation cannot be ascribed to reduced emotional reactivity or anxiety, and may thus relate principally to dysfunctions in central autonomic regulation. NHMRC (Australia)
Effects of exercise on miRNA expression levels in human peripheral blood mononuclear cells (PBMCs)
Authors: Radom‐Aizik Shlomit , Zaldivar Frank , Oliver Stacy , Galassetti Pietro , Cooper Dan M
Published: June 2021

Brief exercise results in substantial changes in gene expression of circulating PBMCs. Moreover, many key gene pathways influenced by exercise are related to inflammation. We hypothesized that microRNAs (small 21–23 nucleotide non‐coding RNAs) would also be changed by exercise and act to prevent a predominantly pro‐inflammatory PBMC response that could be dangerous. 12 healthy men (19–30 y.o.) performed 20 min of cycle ergometry (~75% peak VO2). PBMCs were isolated before and after the exercise. Using the Agilent Human miRNA Microarray (V2), we found that exercise altered 94 miRNAs (FDR=0.2; 61 downregulated). Many of the miRNAs altered by exercise are known to play mechanistic roles in other inflammatory processes [e.g., miR‐125b(↓) and miR‐146a(↓) which are downregulated by proinflammatory factors LPS and TNF‐α, respectively). We also found exercise effects on miRNAs associated with B‐cell development, e.g., miR‐181a(↑), ‐150(↓), and ‐17(↓). Further, we compared the miRNA changes in PBMCs with the 38 miRNAs in neutrophils that we previously found to have been altered by exercise. 18 miRNAs were affected in both PBMCs and neutrophils, but only 12 changed in the same direction. In conclusion, brief heavy exercise changes miRNA profiles in PBMCs many of which are related to inflammatory processes. The pattern of change suggests that exercise differentially influences miRNAs in leukocyte subtypes.

Grants: Supported by NIH grants P01‐HD048721 and RO1‐HL080947