bluuebunny/crossref_metadata_2025 huggingface dataset:
Calorie restriction as a new approach for resetting carotid body chemoreflex control in heart failure (873.11)
Authors: Del Rio Rodrigo , Marcus Noah , Schultz Harold
Published: June 2021

The carotid body (CB) plays a pivotal role in the increased chemosensitivity following chronic heart failure (CHF). The mechanisms underlying the CB potentiation in CHF are not fully understood. However, it is known that oxidative stress contributes to the exaggerated CB function. Since calorie restriction (CR) has been shown to reduce oxidative stress we hypothesized that CR normalizes CB afferent activity and restores normal chemoreflex drive in CHF. Male rats (200g) were subjected to coronary artery ligation or sham surgery. CHF and sham animals were randomly allocated into 2 groups: Ad libitum calorie intake (AL) or CR (30% less caloric intake compared to AL). Ventilatory chemoreflex responses to hypoxia (FiO2~10%) were measured 4 weeks after AL or CR. CB chemoreceptor afferent neural activity was recorded and Mn‐superoxide dismutase (SOD2) protein expression was assessed in CB chemoreceptor cells. CHF‐AL rats compared to sham‐AL rats displayed exaggerated ventilatory chemoreflex response to hypoxia, potentiation of CB afferent discharge, and reduced SOD2 expression in CB chemoreceptor cells (21±1 vs. 73±3 a.u., CHF‐AL vs. sham‐AL, p<.05). CR reduced ventilatory chemoreflex function in CHF rats to levels comparable to the ones obtained in sham‐AL rats. CR in sham rats had no effect on chemoreflex function. Compared to CHF‐AL rats, CHF‐CR rats displayed reduced CB afferent discharge during hypoxia (150±13 vs. 262±21 Hz, CHF‐CR vs. CHF‐AL, p<.05) and a 3‐fold increase in SOD2 protein expression in CB chemoreceptor cells. Our results show that CR increases SOD2 expression in CB chemoreceptor cells, prevents abnormal CB afferent function, and normalizes chemoreflex control in CHF rats.

Grant Funding Source: Supported by NIH PO1‐HL62222
Unilateral renal denervation decreases adrenergic and angiotension II type I receptors in the PVN of rabbits with chronic heart failure (875.15)
Authors: Schiller Alicia , Haack Karla , Zucker Irving
Published: June 2021

Hallmarks of chronic heart failure (CHF) are increased sympathetic tone and blunted baroreflex sensitivity. Renal denervation (DNx) improves these symptoms in hypertensive patients and in experimental models of CHF. However, the mechanism(s) underlying the protective effects of renal DNx are unknown. The paraventricular nucleus (PVN) is a cardiovascular control region of the brain known to influence all sympathetic nerve beds. Both α‐ and β‐adrenergic (α1cAR, β1AR) and angiotensin II type 1 (AT1) receptors in the PVN contribute to baroreflex sensitivity. Changes in α1cAR, β1AR and AT1 expression in the PVN following renal DNx are unclear. We hypothesized that renal DNx would attenuate the adrenergic and AT1 alterations in the PVN in CHF. 13 New Zealand White rabbits either underwent DNx (stripping the left renal artery of all visible fibers) / sham(INV) surgery. Four weeks post‐surgery, animals were then either not paced (Sham‐INV), or paced at 380 BPM to induce CHF (CHF‐INV or CHF‐DNx), which was confirmed by echocardiography. Western blot analyses of PVN micropunch lysates indicated that CHF‐INV rabbits had a significant increase in β1AR compared to Sham‐INV (0.17 ± 0.01 vs 0.11 ± 0.02, p < .05) and this was attenuated in CHF‐DNx rabbits (0.09 ± 0.001, p < .05). Compared to CHF‐INV rabbits, CHF‐DNx rabbits showed a decrease in both α1cAR (0.56 ± 0.17 vs 0.23 ± 0.01) and AT1 (0.38 ± 0.09 vs 0.15 ± 0.03) protein. These results suggest that withdrawal of the renal nerves by DNx decreases receptors within the PVN that impact sympathetic outflow in CHF.

Grant Funding Source: Supported by AHA PREDOC14700045 and PO1HL62222
The effects of ketamine on the immune response in the fetal ovine brain in response to hypoxic hypoxia (877.8)
Authors: Thomas Brandi , Chang Eileen , Miguel Zarate , Arndt Thomas , Wood Charles
Published: June 2021

Fetal hypoxia is a reduction in oxygen supply to the fetus. HH occurs when maternal and fetal partial pressure of oxygen is decreased due to maternal partial ventilatory restriction. Recently, our lab reported that HH increased inflammatory genes in the fetal brain, and KET reduced the expression by microarray analysis. We hypothesized that HH causes an inflammatory reaction in the fetal ovine brain, and KET attenuates this response. We examined fetal ovine brain (n=3‐5/group) where mother and baby were chronically catheterized, and tracheostomy was performed on the ewe. A 50 % decrease in partial pressure of oxygen was induced by administering nitrogen gas directly to the ewe for 30 min. KET was administered intravenously to the fetus 10 min prior to the induction of HH. Brains were collected 24 hrs post HH stimulus, fixated in 4% paraformaldehyde, and then embedded in paraffin blocks for hematoxylin and eosin staining. After tissue examination under light microscope, lymphocytic infiltration, congestion in blood vessels, and the presence of proteinaceous substances were evaluated by a 5‐point scoring criteria in the HH brains. KET produced a decrease in inflammatory response in tissues subjected to HH, and the induction of HH produced a higher inflammatory response when compared to normoxia group (P<0.05, Friedman test). We conclude that KET decreases the level of inflammation in the fetal ovine brain exposed to HH.

Grant Funding Source: Supported by NIH grants HD033053 and HD05761 and by the APS STRIDE program.
Constitutive nitric oxide synthase gene and protein expression in the green anole lizard (Anolis carolinensis) (879.18)
Authors: Forgan Leonard , McNeill Bryony , Edwards Susan , Donald John
Published: June 2021

Vertebrate metabolic homeostasis is integrally linked to the maintenance of blood flow, a process arising from a complex interplay between neural and humoral factors. A key effector in this process is nitric oxide (NO). The constitutive nitric oxide synthase (NOS) isoforms, NOS1 and NOS3, are involved in NO production in mammalian blood vessels, but this is not the case in vertebrates in general. Previous research has shown that the NO control of vertebrate blood vessels has undergone a series of key evolutionary steps. For example, NO vasodilation first appears in some teleost fish but is ubiquitous in tetrapods. Interestingly, NOS 1 is expressed in perivascular nerves of many vertebrates including fish. However, the NOS3 gene first appears in amphibians but is not expressed in the vascular endothelium. Furthermore, NOS3 can be identified in reptilian and avian genomes. Thus, the question then arises as to when did the endothelium first express the NOS protein. The aim of this research is determine if reptiles were the first vertebrates to express NOS3 in the vascular endothelium. We have obtained a full clone of a nos3 cDNA and a partial clone of nos1 from the green anole, Anolis carolinensis. The deduced NOS3 protein from the cDNA clone varies from the NOS3 protein predicted from the Anolis genome. We will present in situ hybridisation data using fully homologous constitutive NOS riboprobes and immunohistochemical data using a custom antibody for the A. carolinensis NOS3 protein.
Plasticity of adenosine and endothelin I tone on cardiovascular function in embryonic chickens (Gallus domesticus): effects of developmental hypoxia (879.5)
Authors: Crossley Dane , Iversen Nina , Baatrup Erik , Wang Tobias
Published: June 2021

Chronic hypoxic incubation of embryonic chickens is a common environmental challenge used to assess plasticity in the development of cardiovascular function in vertebrates. Hypoxic stress increases vascular growth, circulating catecholamines, and cholinergic tone while decreasing resting arterial pressure, adrenergic tone, and sensitivity to exogenous nitric oxide in some chicken breeds. Given the limited contribution of the central nervous system to cardiovascular homeostasis throughout the majority of chicken development hypoxia may also alter regulatory elements if they exhibit plasticity. We investigated the plasticity of key local regulatory ligands, adenosine and endothelin I in embryonic chickens induced by hypoxia (15%O2). At day 15 (70%) and 19 (90%) of a 21 day incubation period embryos from normoxic and hypoxic conditions were instrumented to measure arterial pressure and heart rate. Adenosine produced a dose dependent hypotensive bradycardia that increased in intensity with age. Endothelin I produced a pronounced hypertension at the maximal dose on day 15 and a dose dependent hypertension at day 19. Chronic hypoxic incubation blunted the adenosine response while it intensified the pressure response to endothelin I. Finally, the embryonic cardiovascular response adenosine receptor blockade with theophylline on day 19, a sustained hypertension, was absent in embryos incubated in 15% O2. These findings indicate embryonic chickens utilize adenosine and endothelin I to maintain cardiovascular homeostasis. Further both responses exhibit regulatory factor specific plasticity.

Grant Funding Source: Supported by NSF CAREER IBN IOS‐0845741 to DAC
Oxidative stress impairs vasodilation of ex vivo arteries from mourning doves (Z. macroura) (880.3)
Authors: Sweazea Karen , Jarrett Catherine , Faust James
Published: June 2021

In mammals, high plasma glucose concentrations (PGlu) lead to oxidative stress‐induced endothelial dysfunction and impaired vasodilation. However, avian PGlu are normally 1.5‐2 times greater than mammals of similar size. Therefore, the hypothesis was that endothelium‐dependent vasodilation of isolated avian arteries would not be impaired following acute exposure to high glucose. Isolated small resistance cranial tibial arteries (80‐150μm, inner diameter) were cannulated and pressurized in a vessel chamber then incubated with a physiological solution containing either normal or high glucose (20mM vs. 30mM) for 1h at 41°C. Vessels were then pre‐constricted to 50% of resting inner diameter with phenylephrine (PE) and exposed to increasing doses of acetylcholine (ACh; 10‐9 to 10‐5 M, 5 mins per step). Percent reversal of PE‐induced tone was measured by tracking the inner diameter with edge‐detection software. Contrary to our hypothesis, ACh‐induced vasodilation was impaired with acute exposure to high glucose (p=0.013). The impairment was not related to increased osmolarity since vasodilation of arteries exposed to a combination of 20mM glucose and 10mM mannitol was not different from controls (p=0.898). Superoxide concentrations were significantly increased in arteries exposed to high glucose (168 ± 42%, p = 0.0072) and pre‐exposure of arteries to the superoxide dismutase mimetic tiron (10mM) improved vasodilation (p<0.05). Therefore, dove arteries are subject to oxidative stress‐mediated pathologies in conditions mimicking hyperglycemia.
Effect of posture on heart rate control prior to and following dynamic leg exercise (881.8)
Authors: White Daniel , Kay Victoria , Moralez Gilbert , Benson Tyler , Carlton Joshua , Eubank Wendy , Raven Peter
Published: June 2021

Reflex control of cardiovascular function is paramount in maintaining homeostasis both before and after exercise. We investigated the influence of posture on arterial baroreflex function both before and after prolonged moderate to high intensity exercise.

9 healthy subjects (1 female) aged 24.4 years (±1.16) volunteered for protocol 1 of the study and 13 healthy subjects (3 female) aged 25.5 years (±0.88) volunteered for protocol 2. Each subject underwent graded exercise test either upright (UR) (protocol 1) or semi‐recumbent (SR) (protocol 2). During both protocols 1 and 2 neck pressure/neck suction protocols were performed at rest, exercising HRs of 120 and 150 bpm, and recovery. Reflex data for HR and MAP were analyzed and baroreflex curves were calculated. Repeated measure ANOVAs was performed and Neuman‐Keuls post hoc analysis was used when differences were detected. Significance was set at P<0.05.

Significant differences were identified in operating point values of HR between SR and UR at rest (P<0.001), and between rest and recovery in both SR and UR (p<0.001). Differences were also identified in Carotid‐cardiac baroreflex response range and maximum gain between and within all conditions (p<0.05).

Differences in the neural reflexive control of HR at rest and during recovery are related to postural changes in cardiopulmonary loading.

Grant Funding Source: Supported by TACSM SRDA
Breathing heliox reduces expiratory flow limitation and improves exercise performance in adult survivors of extreme preterm birth (882.4)
Authors: Duke Joseph , Mangum Tyler , Elliott Jonathan , Gladstone Igor , Lovering Andrew
Published: June 2021

Extreme preterm birth (<32 weeks gestational age) results in arrested development of the lung characterized by abnormal alveologenesis and pulmonary vasculogenesis causing obstructive lung disease that persists into adulthood. We have recently shown that adults with a history of extreme preterm birth (PRE) have reduced aerobic exercise capacity relative to full term controls (CONT) that is not explained by pulmonary gas exchange inefficiency during exercise. These PRE subjects also have significant exercise‐induced flow limitation (EIFL), which has been suggested to be associated with a reduction in exercise capacity in healthy humans and in COPD subjects. Therefore, we hypothesized that reducing EIFL by breathing heliox (21% O2, 79% He) would improve exercise performance. Both PRE (n = 4) and CONT (n = 4) subjects performed two cycle ergometer exercise tests to exhaustion at 80% of their maximum power output; one breathing room air and one breathing heliox. Measurements breathing heliox were compared to those breathing room air at iso‐time. Flow‐volume loops were measured at rest and during both exercise tests to quantify EIFL. EIFL significantly decreased in PRE from room air (57% of tidal volume; VT) to heliox (9% VT), while CONT did not exhibit EIFL in either condition. Time to exhaustion significantly increased in PRE (38%), but not CONT (9%), breathing heliox compared to room air. These data suggest that excessive EIFL, may lead to an excessive work of breathing and/or mechanical constraints to increasing tidal volume during exercise, thereby significantly limiting exercise performance in PRE subjects.

Grant Funding Source: Supported by AHA Scientist Development Grant and OHSU Early Clinical Investigator Grant
Sleep efficiency associated with nocturnal blood pressure and heart rate dipping in young adults. (883.1)
Authors: Ross Amanda , Yang Huan , Larson Robert , Carter Jason
Published: June 2021

Short sleep and fragmented sleep are associated with blunted nocturnal blood pressure (BP) dipping in humans. Despite epidemiological evidence that blunted nocturnal heart rate (HR) dipping is an independent risk factor for all‐cause mortality, the link between sleep efficiency and nocturnal HR dipping remains unclear. We hypothesized that subjects with low sleep efficiency (<85%) would have blunted nocturnal BP and HR dipping compared to subjects with high sleep efficiency (>85%). Twenty‐two normotensive subjects (13 men, 9 women; age 18‐28 years) wore an Actigraph watch for 7 days and nights, and an ambulatory BP monitor for 24 hours. There were no differences in age, sex, body mass index, mean sleep time, number of awakenings, or 24‐h blood pressure between the low (n=12) and high (n=10) sleep efficiency groups. However, the low sleep efficiency group demonstrated blunted dips in nocturnal systolic BP (9.9% vs. 14.2%, P=.04) and HR (12.4% vs. 21.4%, P=.03), as well as higher mean nocturnal HR (62.5 vs. 54.7 beats/min, P=.02), when compared to the high sleep efficiency group. In conclusion, our findings support growing evidence that sleep efficiency, independent of total sleep time, may be an important cardiovascular risk factor.
Heat balance in SHR is impaired during exercise until fatigue in warm environment (884.1)
Authors: da Cunha Daise , Campos Helton , Leite Laura , Drummond Lucas , Coimbra Cândido , Natali Antônio , Prímola‐Gomes Thales
Published: June 2021

Heat balance in SHR is impaired during exercise until fatigue in warm environment Daise N.Q. Cunha1, Helton O. Campos1, 3, Laura H.R. Leite2, Lucas R. Drummond1, Cândido C. Coimbra3, Antônio J. Natali1, Thales N. Prímola‐Gomes1. Department of Physical Education, UFV, Viçosa, Brazil1. Department of Physiology, UFJF, Juiz de Fora, Brazil2. Department of Phvsiology and Biophysics, UFMG, Belo Horizonte, Brazil3.

To investigate thermal balance in SHR subjected to submaximal exercise until fatigue (SMEF) in warm environment. Male SHR and Wistar rats (WIS) were submitted to SMEF at 60% VO2max and 5º inclination in thermoneutral (25°C, TE) and warm (32°C, WE) environments. Body (Tb) and tail skin (Ttail) temperatures, and oxygen consumption (VO2), were registered during exercise. Total time of exercise (TTE), workload (W), mechanical efficiency (ME) and heat storage (HS) were determined. SHR‐WE showed higher heat production in comparison to WIS‐WE (from 15‐17 min). Baseline Ttail was greater in WE, but demonstrated an equal increase among SHR and WIS in both environments. Animals exercising in the WE had higher Tb in comparison to TE (from 4‐17 min), while SHR‐WE showed higher Tb in comparison to SHR‐TE after 8 min of exercise. SHR had lower ME compared to WIS in TE and WE. Moreover, SHR‐WE showed higher HS compared to SHR‐TE. In conclusion SHR exhibited an increase in heat production and heat storage during exercise until fatigue in warm environment resulting in aggravated hyperthermia linked to lower mechanical efficiency. We used FASEB Statement of Principles for the use of animals in research.

Grant Funding Source: CAPES
A single bout of moderate intensity resistance exercise improves neural mechanisms of arterial pressure control in L‐NAME hypertensive rats (884.14)
Authors: Barreto André , Mota Marcelo , da Silva Tharciano , Fontes Milene , Santana‐Filho Valter , Santos Márcio
Published: June 2021

Resistance training has been used as non‐pharmacological approach for control hypertension. A single bout of exercise can lead to a post exercise decreased blood pressure especially in hypertensive individuals. However neural central mechanisms involved in this modality of exercise remain unclear. Therefore the aim of this study was to evaluate the acute effects of a single bout of resistance exercise (RE) on blood pressure, baroreflex sensitivity (BRS) and cardiac autonomic balance in L‐NAME hypertensive rats. The resistance exercise (RE) consisted of 10 sets of 10 repetitions with 1 minute of interval between sets performed at 60% of one repetition maximum test, in squat‐training apparatus. Mean arterial pressure (MAP) was measured by a catheter placed in the carotid artery and recorded 15 before (baseline) and during 120 minutes post exercise. BRS was analyzed by sequence method and cardiac autonomic balance by heart rate variability in the frequency domain. Hypertension decreased BRS (from 2.03 ± 0.3 to 1.05 ± 0.2 mmHg/s, p<0.05) and increased both LF/HF ratio (from 0.12 ± 0.03 to 0.34 ± 0.05 nu, p<0.01) and LFsys (from 2.69 ± 0.1 to 5.19 ± 0.5 mmHg2, p<0.05) in baseline time. A single bout of moderate RE reduced MAP between 45’ to 120’ (p<0.001) and heart rate (HR) from 75’ to 120’ (p<0.05). Furthermore increased BRS from 15’ to 105’ (p<0.01) and both LF/HF ratio (p<0.05) and LFsys (p<0.001) were reduced. These results suggest that a single bout of moderate RE promotes a better hypertension control by improved BRS and cardiac autonomic balance in L‐NAME hypertensive rats.

Grant Funding Source: Supported by FAPITEC/SE
Metabolic profile and inflammatory markers in an endurance trained and untrained population (884.20)
Authors: Vera Desiree , Castillo Micaela , Oliver Kari , Rosario Emily , Granquist Megan , Dunn Sarah
Published: June 2021

Obesity is reaching epidemic proportions worldwide. Studies have now shown that obesity is the result of an imbalance in metabolic factors that have been triggered by a physiological stress resulting in an inflammatory state. Physical activity may positively impact metabolic dysfunction (insulin, glucose) and inflammatory markers, C‐reactive protein (CRP) and Interluekin‐6 (IL‐6), in adults, ultimately influencing one's risk for disease. With training, IL‐6 has been shown to elicit a pro‐ or anti‐inflammatory like state within the muscle; therefore it is a multi‐factorial cytokine. Whether there is a significant difference between endurance trained and untrained male and female adults in association to metabolic profile and inflammation markers is unknown. Therefore, the purpose was to examine the differences between body composition and metabolic profile (insulin, CRP, IL‐6, etc.) in trained (n=15, age 26.9 ± 14.4 yr) and untrained (n=19, age 36.7 ± 19.2 yr) adults. Fasting venous whole blood (40 ml) and body composition measurements (percent body fat, fat mass, etc.) were obtained from apparently healthy individuals. Enzyme‐linked immunosorbent assays were conducted to analyze fasting blood plasma concentrations of insulin, CRP, and IL‐6. An Independent T‐test was used to identify significant differences (p < 0.05) between trained and untrained (N=35). Significant differences exist in body composition (p < 0.05) and CRP (p < .001). Although fasting plasma insulin and IL‐6 were elevated in the untrained group, no significant differences were found. In conclusion, a significant difference exists between trained and untrained individuals for body composition and CRP suggesting that physical activity may decrease one’s risk of developing disease. Despite a significant difference with CRP in the trained versus untrained individuals, IL‐6 may elicit an anti‐inflammatory like property with endurance training ultimately protecting the trained individuals.

Grant Funding Source: Supported by STEM
Effects of treadmill exercise on spatial learning memory and upregulation of reelin signaling pathway in autistic rats (884.29)
Authors: Baek Seung‐Soo , Ji Eun‐Sang , Lee Min‐Hee
Published: June 2021

Reelin is an extracellular glycoprotein that is essential for neuronal migration and brain development. Neuroprotective effects of exercise on various brain insults are well documented, however, the effects of exercise on autism in relation with reelin expression are not clarified. In the present study, we investigated the effects of treadmill exercise on the functional recovery and on the expressions of reelin and its downstream molecules, phosphatidylinositol‐3‐kinase (PI3K), phosphorylated Akt (p‐Akt), phosphorylated extracellular signal‐regulated protein kinase 1 and 2(p‐ERK1/2), using autistic rats. The rat in the treadmill exercise groups were forced to run on a treadmill for 30 min once a day, five times a week for 4 weeks, starting postnatal day 28. To investigate autism‐like behaviors and memory deficit, open field, social interaction, and radial 8‐arm maze were performed. In the present results, treadmill exercise alleviated aggressive tendency and improved correct decision in the spatial learning memory in the autistic rats. Treadmill exercise increased neurogenesis and the expressions of reelin and its down‐stream molecules, PI3K, p‐Akt, and p‐ERK1/2, in the hippocampus of the autistic rats. The present study showed that treadmill exercise ameliorated aggressive behavior and improved spatial learning memory through activation of reeling signaling pathway in the valproic acid‐induced autistic rats.

Grant Funding Source: This work was supported by the National Research Foundation
Exercise training combined with insulin attenuates cardiac mitochondrial dysfunctions in diabetic rats (884.9)
Authors: da Cunha Daise , da Silva Márcia , Natali Antonio , da Silva Edson , Teodoro Bruno , Drummond Lucas , Drummond Filipe , Moura Anselmo , Belfort Felipe , Oliveira Alessandro , Prímola‐Gomes Thales , Maldonado Izabel , Alberici Luciane
Published: June 2021

Exercise training combined with insulin attenuates cardiac mitochondrial dysfunctions in diabetic rats Daise N.Q. Da Cunha1, Márcia F. da Silva3, Antonio J. Natali2, Edson da Silva3, Gilton G. Jesus2, Bruno G. Teodoro4, , Lucas R. Drummond2, Filipe R. Drummond2, Anselmo G. Moura2, Felipe G. Belfort2, Alessandro Oliveira5, Thales N. Prímola‐Gomes2, Izabel R.S.C. Maldonado3, Luciane C. Alberici4. Physical Education Department1, Veterinary Medicine Department2, General Biology Department3, Physics and Chemistry Department4, Nutrition and Health Department5. 1,2,3, and 5UFV, Viçosa, and 4USP, Ribeirão Preto, Brazil

Type 1 diabetes mellitus (T1DM) leads to cardiac mitochondrial dysfunction. We tested whether aerobic exercise training combined with insulin therapy attenuate the deleterious effects of T1DM on the cardiac mitochondrial function. T1DM was induced by Streptozotocin (IP, 60 mg/kg) in male Wistar rats. They were submitted to swimming training (90 min/day, 5 days/week, 5% body mass) and received daily insulin, 4 IU, for 8 weeks. Mitochondrial function was assessed in isolated cardiac mitochondria. Diabetes increased mitochondrial Ca2+ uptake and the susceptibility of the mitochondrial permeability transition pore (MPTP) opening, augmented resting respiration rates and reduced mitochondrial H2O2 release. Exercise training and insulin, singly, reduced the mitochondrial Ca2+ uptake; while the combination of both restored the susceptibility of MPTP opening, the rates of O2 consumption and H2O2 release in diabetic rats. In conclusion, aerobic swimming training associated with insulin therapy attenuated the disruptions of mitochondrial Ca2+ uptake, respiration rates, and H2O2 release in isolated cardiac mitochondria of rats with T1DM. We used FASEB Statement of Principles for the use of animals in research.

Grant Funding Source: FAPEMIG
The crucial role of calcium sensing receptor for hypoxia‐induced pulmonary arterial hypertension in mice (889.2)
Authors: Tang Haiyang , Song Shanshan , Drennan Abigail , Fernandez Ruby , Chen Jiwang , Yuan Jason X.‐J.
Published: June 2021

Rationale: An increase in cytosolic free Ca2+ in pulmonary arterial smooth muscle cells (PASMC) is not only a major trigger for pulmonary vasoconstriction but also an important stimulus for PASMC proliferation. Our previous data showed that the extracellular calcium sensing receptor (CaSR) expression and function were both enhanced in PASMC from idiopathic PAH patients. However, the implication of CaSR in the development of PAH remains elusive.

Methods: CaSR and parathyroid hormone double knockout (DK) mice were used to test the hypothesis that CaSR modulates pulmonary vascular tone and the response to hypoxia. Wild type (WT) and DK mice were exposed to 10% oxygen for up to three weeks. Pulmonary vascular remodeling was evaluated using tissue morphometrics. Right ventricle systolic pressures (RVSP) and right ventricular hypertrophy by, RV/(LV+S) ratios, were measured to evaluate pulmonary hypertensive development. We also isolated PASMC from WT or DK mice, followed with cell proliferation assay and cytosolic Ca2+ concentration ([Ca2+]cyt) measurement.

Results: WT and DK mice displayed normal systemic and pulmonary arterial pressure under normoxic conditions. However, compared to WT mice, DK mice had significantly lower RVSP, RV/LV+S ratios, and displayed less pulmonary vascular remodeling when exposed to hypoxia. Extracellular Ca2+ caused an increase in [Ca2+]cyt in PASMC isolated from hypoxic WT mice. However, restoration of extracellular Ca2+ had little effect on [Ca2+]cyt in DK mice. Additionally, a decrease in store‐operated Ca2+ entry (SOCE) was observed in PASMC from DK mice, as compared to SOCE in PASMC from WT mice.

Conclusion: These data indicate that CaSR plays an important role in the development and progression of pulmonary vascular remodeling in mice with PH. CaSR deletions prevent the development of hypoxia‐induced PAH in mice via its modulation of extracellular Ca2+ entry in PASMC, suggesting that CaSR may be a novel therapeutic candidate for the treatment of PAH.

Grant Funding Source: Supported by NIH (HL066012; HL115014)
Pre‐existing hypoxia sensitizes the kidney to an ischemia‐reperfusion insult (890.10)
Authors: Pihl Liselotte , Nangaku Masaomi , Inagi Reiko , Liss Per , Palm Fredrik , Nordquist Lina
Published: June 2021

Hypoxia plays an important role in progression of kidney disease. Indeed, diabetic kidney develops pronounced hypoxia and is sensitive to an ischemia‐reperfusion (IR) insult. However, diabetes is also characterized by hyperglycemia and oxidative stress. It is therefore important to delineate the role hypoxia per se. We therefore developed a protocol to induce intrarenal tissue hypoxia independently of the diabetic condition using the mitochondrial uncoupler dinitrophenol (DNP).

Rats with and without DNP‐induced hypoxia were subjected to 40 min of kidney ischemia and function measured 4 weeks thereafter.

IR to DNP‐treated rats resulted in 30% lower glomerular filtration rate, whereas it did not affect kidney function in untreated rats.

The aim of this project was to investigate if pre‐existing tissue hypoxia sensitizes the kidney to an IR insult independently of hyperglycemia and oxidative stress.

Pre‐existing intrarenal hypoxia sensitizes the kidney to an IR insult. It may therefore be beneficial to target kidney hypoxia as a strategy to prevent or reduce the often detrimental effect of an IR insult to the diabetic kidney.
Hyperaldosteronism in KCa1.1 channel β2‐subunit KO mice (892.15)
Authors: Larsen Casper , Sorensen Mads , Praetorius Helle , Leipziger Jens
Published: June 2021

Whole body K+ homeostasis depends on matching K+ excretion to the daily K+ intake. K+ is excreted via the colon and the kidney, the latter handling the majority of the total K+ excretion. Aldosterone is an important regulator of both renal and colonic K+ excretion. Renal K+ secretion depends on 2 apical K+ channels, ROMK and KCa1.1, while colonic K+ secretion depends on apical KCa1.1 channels. We have acquired a novel global knockout mouse for the β2‐subunit of the KCa1.1 channel, which is the only KCa1.1 β‐subunit expressed in murine colonic epithelium.

The β2 KO mice have increased plasma aldosterone. Primary hyperaldosteronism is characterized by low plasma K+, low renin and hypertension. β2 KO mice have normokalemia and low renin, indicating a secondary cause of hyperaldosteronism. The low renin in β2 KO mice indicates that a K+ handling deficiency, rather than hypotension, is causing the hyperaldosteronism.

BK channel‐dependent colonic K+ secretion was not disturbed in β2 KO mice In contrast, urinary K+ excretion was reduced in β2 KO mice (P=0.06, n=5), suggesting that β2 KO mice have a reduced capacity for renal K+ secretion. mRNA expression of the β2 subunit has previously been found in rabbit cortical collecting ducts, where expression is increased by K+ loading and reduced by K+ depletion. These data indicate an important function of the β2 subunit of the KCa1.1 in renal K+ excretion.

Grant Funding Source: Supported by The Lundbeck Foundation
Acute inhibition of calcineurin using cyclosporine activates renal salt transporters (892.31)
Authors: Blankenstein Katharina , Borschewski Aljona , Ellison David , Bachmann Sebastian , Mutig Kerim
Published: June 2021

Renal tubular transport processes are essential for systemic water and salt homeostasis and blood pressure control. The renal distal cation‐chloride cotransporters NKCC2 and NCC play a pivotal role in volume regulation. Increased activity of these transporters may cause salt retention and hypertension. Both NKCC2 and NCC are activated by N‐terminal phosphorylation. We recently showed that chronic administration of a calcineurin inhibitor tacrolimus was associated with increased NCC phosphorylation suggesting a role of the phosphatase in deactivation of the distal transporters. In this study we test acute effects of another calcineurin inhibitor, cyclosporine A (CsA) on NKCC2 and NCC in vivo and in cultured cells. The expression of cyclophilins, interacting with CsA during calcineurin inhibition, has been localized to the distal nephron using in situ hybridization and immunohistochemistry. Administration of CsA (25 mg/kg) to normal rats for 4 h led to marked increases of phosphorylated NKCC2 (3 fold) and NCC (6 fold). The concomitant analysis of total NKCC2 or NCC abundance revealed no significant changes. To exclude potential systemic influences, cultured rat TAL cells were treated with CsA for 4 h, which led to a substantial increase of NKCC2 phosphorylation. Our data suggest that phosphorylation of NKCC2 and NCC is acutely regulated by CsA at posttranscriptional level. Considering the broad clinical application of CsA and its renal side effects such as hypertension and electrolyte disorders, understanding of mechanisms of its side effects has clinical implications.

Grant Funding Source: DFG 88126042
Aldosterone enhances urine flow in mice on a low Na, high K diet (892.7)
Authors: Cornelius Ryan , Wen Donghai , Sansom Steve
Published: June 2021

Mice on a low Na, high K diet (LNaHK) are able to excrete excess K and maintain K homeostasis by increasing tubular fluid flow in the distal nephron of the kidney. High tubular fluid flow increases K secretion by activating BK‐α/β4 channels in the collecting duct, which are regulated by aldosterone (aldo). The effect of aldo on tubular fluid flow has not yet been investigated. We hypothesized that aldosterone enhances tubular fluid flow in mice on LNaHK. C57BL/6 mice underwent adrenalectomy (ADX) or sham surgery and were given either LNaHK or control diet. ADX LNaHK received aldo replacement, via osmotic pump, at low dose (L‐Aldo; analogous to sham control diet mice) or high dose (H‐Aldo; analogous to sham LNaHK mice). Plasma [aldo] was higher in L‐Aldo (728 pg/ml) compared to sham control diet mice (287 pg/ml), while H‐Aldo (1754 pg/ml) was lower than sham LNaHK (3630 pg/ml). L‐Aldo exhibited a urine flow (UV) of 1.4 ml/day, a UKV of 1005 μmol/day, and a luminal [K] of 125 mM. H‐Aldo demonstrated an increased UV and UKV of 5.0 ml/day and 2714 μmol/day, respectively, whereas the luminal [K] was not significantly different (125 mM). The results demonstrate that L‐Aldo enhances K secretion in mice on LNaHK by increasing K secretion in the distal nephron, while H‐Aldo further increases K secretion by enhancing distal flow. This indicates that high levels of aldo is needed in mice on LNaHK to enhance UV enough to excrete the excess K.

Grant Funding Source: Supported by AHA Predoctoral Fellowship 11PRE7530018
TRPV4 deficiency protects myocardium following myocardial infarction and transverse aortic constriction (893.11)
Authors: Cappelli Holly , Adapala Ravi , Thoppil Roslin , Ohanyan Vahagn , Luli Jordan , Luther Daniel , Paruchiri Sailaja , Meszaros J. Gary , Chilian William , Thodeti Charles
Published: June 2021

Cardiac remodeling is triggered by a cascade of events following myocardial injury, including the differentiation of cardiac fibroblasts (CFs) into myofibroblasts. Myofibroblasts are hypersecretory and highly contractile cells, however, in excess, can lead to cardiac dysfunction and eventual heart failure. Recently, our lab has shown, in vitro, that TRPV4, a mechanosensitive ion channel, integrates mechanical and soluble signals to aid in the differentiation process of CFs. Our current study aims to investigate physiological role of TRPV4 during the remodeling process by subjecting wild type (WT) and TRPV4 knockout (KO) mice to myocardial infarction (MI) or transverse aortic constriction (TAC). Assessment of the post‐surgery survival and cardiac function found the TRPV4 KO mice to have improved survival rates and preserved ejection fraction. Cardiac fibrosis was examined using picrosirius red staining which revealed that the TRPV4 KO mice had significantly less fibrosis than their WT counterparts. Furthermore, a comparison of gene expression in the whole heart tissue allowed analysis of pro‐fibrotic genes, such as col1A2, α‐SMA, N‐FAT, TGF‐β1, and mechanosensitive transcription factor MRTF‐a, and found a reduction in the expression of these genes in the TRPV4 KO mice pre‐ and post‐cardiac injury. Altogether, our findings suggest that the absence of TRPV4 improves the cardiac function and remodeling following myocardial injury, and could lead to a novel therapeutic target for cardiac fibrosis.

Grant Funding Source: Supported by NIH‐1R15HL106442‐01
Inhibition of KCa3.1 decreases differentiated osteoclast function in RAW264 cells (893.21)
Authors: Jones Heather , Birchard Zackary
Published: June 2021

Human intermediate conductance Ca2+‐activated K+ channels (KCa3.1) are found in many cell types including colonic and airway epithelia, vasculature smooth muscle and T‐lymphocytes. Recent work has shown Ca2+‐activated K+ channels may also be involved in the osteoclast spreading leading to the development of osteoporosis. The aim of this study was to further investigate the role of KCa3.1 in osteoclast differentiation and resorption. Cell culture studies were done using RAW264.7 cells differentiated into active osteoclasts using m‐CSF and RANKL. Initially, KCa3.1 protein was identified in undifferentiated and differentiated RAW264.7 cells using an antibody specific to the KCa3.1 channel. Analysis of the western blots showed similar levels of protein expression in undifferentiated and differentiate osteoclasts. To establish a relationship between KCa3.1 function and osteoclast differentiation, TRAP‐staining was done to visualize differentiated RAW264.7 cells. Cells were treated with m‐CSF and RANKL for five days to differentiate the macrophage cells. Results showed that treatment with DC‐EBIO or clotrimazole (CLT) did not significantly alter the number of differentiated osteoclast. Finally, to elucidate the role of KCa3.1 in osteoclast function a bone resorption assay was used. RAW264.7 cells were differentiated and passed onto an Osteolyse plate containing bone matrix. Cells were treated with CLT and DC‐EBIO and bone resorption was assessed using fluorescence imaging. Treatment of the differentiated cells with CLT at day 7 showed a statistically significant decrease in the resorption and osteoclast function when compared to control differentiated RAW264.7 cells. These results suggest that although KCa3.1 does not play a pivotal role in osteoclast differentiation, activation of the channel may lead to the development of osteoporosis. Moreover, inhibition of the channel showed a decline in osteoclast activity and maybe protective against developing osteoporosis.
Secretory activity of human cyst fluid isolated from polycystic kidney disease patients (893.24)
Authors: Martinez Gabriel , Flaig Stephanie , Blazer‐Yost Bonnie
Published: June 2021

Polycystic kidney disease (PKD) is characterized by the slow growth of fluid‐filled cysts in kidney tubules. Kidney function is relatively normal in the first 5 decades of life despite substantial cyst development but thereafter the decline in function is precipitous leading to complete renal failure in five years in 50% of patients. As cysts increase in size, the probability of rupture and release of cyst fluid becomes increasingly likely. Cyst fluid has been shown to cause an additional secretory Cl‐ flux that leads to the expansion of the remaining intact cysts. We have previously shown that the active component of the cyst fluid is lysophosphatidic acid (LPA). Electrophysiological techniques were used to measure human cyst fluid stimulation of ion flux in the mpkCCDcl4 cell line, a model of the cell type that lines renal cysts. The Cl‐ secretory response is due to activation of both CFTR and a calcium activated chloride channel. Interestingly, the cyst fluid effect was not accompanied by an increase in cAMP but rather via a LPA receptor mediated activation of phospholipase C followed by the stimulation of the tyrosine kinase Pyk. These results suggest novel targets for treatment of late stage PKD.
All‐trans‐retinoic acid ameliorates hepatic steatosis by activating Hes6, a novel transcriptional repressor in mice (901.2)
Authors: Kim Chun‐Ki , Kim Seongchul , Axe David , Cook Aaron , Lee Mikang , Lee Yoon Kwang
Published: June 2021

Mice deficient orphan nuclear receptor Small Heterodimer Partner (SHP) are protected from diet‐induced hepatic steatosis due to increased fatty acid oxidation and decreased lipogenesis. Especially the mice exhibited very low expression of peroxisome proliferator activated receptor gamma 2 (Pparγ2), a potent adipogenic transcription factor, in the liver after long‐term western diet (WestD) regimen. We have identified a hairy and enhancer split 6 (Hes6), a novel transcriptional repressor, as an important mediator for the regulation of Pparγ2 expression by SHP using Illumina Beadchip array and realtime PCR analysis in the RNAs isolated from livers of SHP‐/‐ and SHP‐overexpressing mice. The repression of Pparγ2 expression by Hes6 is achieved via its direct repression of hepatocyte nuclear factor 4 alpha (Hnf4α) transcriptional activity. The Hes6 promoter is specifically activated by the retinoic acid receptor (RAR) in response to its natural agonist all‐trans retinoic acid (atRA), and is repressed by SHP. Treatment of atRA or adenovirus‐mediated RARα overexpression in obese mice increased Hes6 mRNA levels and significantly reduced hepatic fat accumulation along with the decreased expression of Pparγ2 and its downstream target gene, fat specific protein 27 (Fsp27). Furthermore, adenovirus‐mediated Hes6 overexpression significantly reduced triglyceride concentration and the expression of Pparγ2 and Fsp27 in the livers of WestD‐fed mice. The current study identifies a novel transcriptional regulatory cascade controlling lipid metabolism and provides insights into new therapeutic approaches to fatty liver diseases.

Grant Funding Source: This study is supported by NIH grant R01DK093774.
Regulation of intestinal calcium absorption appears to be different in dogs and man (908.9)
Authors: Dobenecker Britta , Schmitt Stephanie , Mack Julia , Kienzle Ellen , Alexander Lucille , Morris Penelope
Published: June 2021

The dog is used as a model for human skeletal research, with the assumption that calcium (Ca) and phosphorus (P) metabolism are similar between species. A prior meta‐analysis of Ca intake and faecal excretion suggested that adult dogs are unable to adapt their Ca digestibility to Ca intake, meaning dogs lack the ability to increase Ca digestibility when dietary Ca is low. We wanted to test this hypothesis in vivo with diets reflective of normal feeding.

Apparent Ca digestibility and selected Ca metabolism and safety markers were determined at baseline and every 6 weeks for 6 months of low Ca feeding (~60 mg Ca/kg BW^0.75 = Minimum Requirement (MR), Ca/P=0.6) in 11 adult dogs. During low Ca feeding there was no decrease in faecal Ca excretion and apparent Ca digestibility was negative. Markers of bone resorption increased. This study confirmed the results of the meta‐analysis: dogs fed Ca at MR exhibit negative apparent Ca digestibility and increased bone resorption suggesting that Ca regulation in the dog does not utilize changes in Ca digestibility. These data show that Ca homeostasis of the dog differs from human, and thus the dog should not be used as a model animal for human skeletal research.
Reproductive effects of phoenixin (911.8)
Authors: Tullock Chloe , Samson Willis
Published: June 2021

Phoenixin is a peptide hormone identified by our group to be produced in the neuroendocrine hypothalamus and pituitary gland (J Neuroendocrinol 25:206,2013). We have demonstrated a direct pituitary action of phoenixin to augment GnRH‐stimulated LH secretion and that compromise of endogenous phoenixin production in hypothalamus delayed the onset of estrus in female rats, suggesting an important role for the peptide in reproductive function. We report here that phoenixin mRNA levels are significantly elevated in hypothalamus and anterior pituitary on diestrous day one when estrogen levels are at their lowest during the rat estrous cycle. In cultures of an immortalized mouse gonadotroph cell line, LβT2, phoenixin treatment for two hours increases Luteinizing Hormone β‐chain mRNA, as determined by real time polymerase chain reaction. Treatment of the cells with estradiol benzoate abrogates this effect. Additionally, ovariectomized animals treated with estradiol benzoate also show a trend toward a decrease in phoenixin gene expression in hypothalamus and anterior pituitary gland. These data demonstrate that phoenixin production and action may be controlled by ambient, gonadal steroid levels, and further support a physiologically relevant role for endogenous phoenixin in the control of gonadotropin release.

Grant Funding Source: SLU President's Research Fund
Efficient in vivo gene transfer to murine renal cells using AAV9 vectors (912.7)
Authors: Konkalmatt Prasad , Asico Laureano , Feranil Jun , Jose Pedro , Armando Ines
Published: June 2021

Adeno‐associated viral (AAV) vectors provide sustained long‐term gene expression with minimal immunological consequences. Therefore AAV vector systems are useful gene‐transfer tools in translational, as well as in basic research studies. Systemic administration of AAV9 vector transduces various tissues efficiently but provides very low transduction in kidney, compared to other organs such as liver, heart, and skeletal muscle. We hypothesized that the administration of AAV9 vector by retrograde infusion via the ureter can provide efficient gene transfer to renal cells and minimize off‐target gene expression. AAV9 vector (1e+11 viral genome particles/100 μl), encoding firefly luciferase (AAVCMVLuc) or eGFP (AAVCMVeGFP) under the control of CMV promoter, was infused into the left kidney via the ureter and held for a period of 15 minutes. Non‐invasive bioluminescence imaging (IVIS‐200, Xenogen Corporation) of the mice treated with AAVCMVLuc after one week showed that the signal was limited to the left kidney. Three weeks following vector administration, the magnitude and distribution of gene expression was measured by luciferase activity assay and immunohistochemistry, respectively. Luciferase activity was 4.6‐fold (n=3, P<0.05) higher in the kidney infused with AAVCMVLuc than the liver. Immunohistochemical analyses of AAVCMVeGFP‐treated mice showed gene expression in renal distal, as well as proximal tubular epithelial cells. These studies show that AAV9 gene transfer via the ureter is a highly efficient method for gene transfer and gene rescue studies in mouse kidney.

Grant Funding Source: 1R01DK090918‐01A1
COLIIA1 overexpression with low level laser therapy after experimental injury (914.5)
Authors: Jacomo Alfredo , Teodoro Walcy , Saleh Samir , Martinez Carlos , Ribeiro Marcelo , Hojaij Flávio , Andrade Mauro , Akamatsu Flávia
Published: June 2021

BACKGROUND: Calcaneous tendon injuries are commonly seen as consequences of sports trauma and costly in terms of surgery and rehabilitation. Low Level Laser Therapy (LLLT) has been used to accelerate healing process and avoid tissue adhesions. We investigated the response of experimental lesions of the calcaneous tendon in rats after LLLT therapy.

METHODS AND MATERIAL: Thirty adult male Wistar rats weighing 250 to 300 g were surgically submitted to bilateral partial transverse section of the calcaneous tendon. The right tendon was treated with LLLT whereas the left tendon served as control. On the third postoperative day, the rats were divided into three experimental groups consisting of ten rats each which were treated with LLLT ( Konf, Aculas ‐ HB 750), 780nm and 80mW for 20 seconds, three times/week for 7, 14 and 28 days. Collagen I, II, and III gene expression were studied.

RESULTS: Increased collagen II gene expression was observed in all experimental groups.

CONCLUSION: mRNA gene expression for COLIIA1 is confirmed with immunolocalization of type II collagen and suggests that it could give support to the interstitial tissues on healing process of the calcaneous tendon. Further studies are needed to confirm LLLT role in tendon healing.