bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Renin-Angiotensin System Activity and Blood Pressure by Vascular Smooth Muscle Cullin-3
Authors: Golosova Daria , Brozoski Daniel , Lu Ko-Ting , Muskus Veitia Patricia , Kumar Gaurav , Sigmund Curt
Published: May 2023

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) in smooth muscle cells (SMCs) cause vascular dysfunction, arterial stiffness, and severe hypertension. We hypothesized that conditional deletion of CUL3 in SMCs causes severe hypertension through a renin-angiotensin system (RAS)-dependent mechanism. Mice carrying a conditional allele of CUL3 were bred to mice expressing a tamoxifen-inducible CRE-recombinase driven by a smooth muscle promoter (ISM-CRE, control). Tamoxifen was administered to generate smooth muscle CUL3 knockout (S-CUL3KO). Primary aortic SMCs were used to elucidate the contribution of CUL3 to intracellular angiotensin II-mediated calcium flux. Calcium response in SMCs was analyzed via ratiometric confocal fluorescent microscopyS-CUL3KO mice demonstrated hypertension following three weeks of tamoxifen, mean arterial pressure (MAP) was 108±1 vs. 140±1 mmHg (ISM-CRE vs. S-CUL3KO). Aorta from S-CUL3KO mice exhibited impaired vasorelaxation to acetylcholine (ACh, max relaxation: 84±4% vs. 16±9%, p<0.05) and sodium nitroprusside (SNP, max relaxation: 95±3% vs. 56±9%, p<0.05). Captopril administration (7 days) markedly reduced MAP to 84±0.3 mmHg and improved vasorelaxation in response to both ACh (72±6%) and SNP (91±4%) in S-CUL3KO mice, suggesting RAS-dependency of the hypertension. Additionally, candesartan treatment (7 days) substantially decreased MAP from 138±1 to 96±3 mmHg suggesting that hypertension in S-CUL3KO group is angiotensin type-1 receptor mediated. Q-PCR and RNAScope analyses demonstrated no difference in renin expression in the kidney between the S-CUL3KO and control mice suggesting a blunting of the baroreceptor mechanism regulating renin mRNA expression. Equilibrium angiotensin II plasma levels in both groups were not different (2517±352 and 2379±549 pmol/L, p>0.05). Preliminary data demonstrated that acute application of 10 μM of angiotensin II to S-CUL3KO SMCs resulted in a sustained increase in intracellular calcium which was abolished by a preincubation with 2 μM of candesartan.Our data suggest that CUL3 may play a vital role in baroreceptor mechanism in the kidney and regulate angiotensin II response in SMCs which are essential for responding to changes in perfusion pressure. Ultimately, understanding CUL3 target proteins may benefit the development of novel treatments for hypertension.

R35 HL144807 (to CDS).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Does inhibition of cytochrome P450 2C9 alter cerebral hemodynamics at rest or during simulated mild hypovolemia?
Authors: Matney Jacob , Buelow Alexander , Skillett Sarah , Ashley John , Song Jiwon , Mixon Chris , Black Christopher , Pereira Hugo , Sprick Justin , Kellawan J.
Published: May 2023

Animal models suggest that cytochrome P450 2C9 (CYP450) plays a critical role in the control of cerebral hemodynamics and autoregulation. However, this observation has not been directly examined in humans PURPOSE: To determine the contribution of CYP450 to cerebrovascular hemodynamics in healthy young individuals at rest and during mild simulated hypovolemia. METHODS: 15 subjects (8 females, tested only during early follicular phase) participated in 1 familiarization and 2 experimental visits. In experimental visits, participants ingested either a CYP450 inhibitor, fluconazole (FLZ 150mg) or a microcrystalline cellulose placebo (PLA 250mg) in randomized, single-blind, crossover design. Following 120 minutes of supine rest after ingestion middle cerebral artery velocity (MCAv, cm/s, Transcranial Doppler), mean arterial pressure (MAP, mmHg, finger photoplethysmography), prefrontal cortex oxygenation (TSI, %, Near-Infrared Spectroscopy) were continuously measured during 5 minutes of supine rest and 5 minutes of lower-body negative pressure (LBNP, -20mmHg). Cerebrovascular conductance index was calculated (CVCi = MCAv/MAP, cm/s/mmHg). Further, gain, coherence, and phase were determined using transfer function analysis of MCAv and MAP data. RESULTS: Resting values for all variables were not different between treatments (p > 0.05). Therefore, all data are presented as a change (Δ) from rest to LBNP ± SD. MCAv decreased from rest to LBNP with FLZ (p = 0.001) but did not differ between treatments (PLA -2.86±6.94 vs. FLZ -6.62±6.32 cm/s, p = 0.18, d = 0.36). Similarly, CVCi decreased between rest and LBNP for FLZ (p = 0.02), but did not differ between treatments (PLA -0.04±0.07 vs. FLZ -0.09±0.13 cm/s/mmHg, p = 0.19, d = 0.35). TSI was unaltered from rest to LBNP, nor by treatment (PLA 0.03±1.45 vs. FLZ -0.02±1.25%, p = 0.91, d = 0.03). Transfer function analysis was performed but data were not interpreted as coherence values were not consistently above 0.5, which is not an unanticipated result when analysis is applied to healthy young adults. CONCLUSION: This study indicates that cytochrome P450 inhibition does not affect middle cerebral artery velocity, CVCi, or prefrontal cortex oxygenation at rest or during mild-hypovolemic stress. Therefore, these data suggest that CYP450 is not compulsory for regulation of cerebrovascular hemodynamics in healthy young adults.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Contribution of cytochrome P450 2C9 pathways to functional sympatholysis in healthy young adults
Authors: Buelow Alexander , Matney Jacob , Skillett Sarah , Ashley John , Song Jiwon , Mixon Chris , Bemben Debra , Larson Daniel , Kellawan J
Published: May 2023

In humans exercise hyperemia is adequately maintained despite elevated sympathetic outflow through an endothelium dependent, yet nitric oxide and prostaglandin independent mechanism(s) known as functional sympatholysis. Evidence suggests that functional sympatholysis has an endothelium-dependent hyperpolarization-like component, however, investigations into hyperpolarization pathways, such as, cytochrome P450 2C9 are scant. PURPOSE: To determine if cytochrome P450 inhibition attenuates functional sympatholysis in healthy young adults. METHODS: 21 participants (23 ± 3 years, 11 Females, tested in early follicular phase) participated in three study visits (1 familiarization, 2 experimental visits, random, single blind, counter-balanced) separated by ≥72 hours. Participants completed 5min baseline, 2min resting lower body negative pressure (LBNP, -20mmHg, to elevate sympathetic outflow), 7min of dynamic handgrip exercise at 20% effort with minutes 5-7 having LBNP stimulus superimposed (Ex+LBNP) in the supine position, exercising limb at heart-level. 10 min of rest separated each event. 120min prior to testing a placebo (PLA, 250 mg microcrystalline cellulose) or cytochrome P450 2C9 inhibitor (150 mg fluconazole, FLZ) were ingested. Forearm blood flow (FBF, Echo and Doppler ultrasound) and mean arterial pressure (MAP, finger photoplethysmography) were measured continuously and use to calculate forearm vascular conductance (FVC= FBF/MAPx100mmHg). RESULTS: All data are mean percent change in FVC (%Δ) ± SD. FVC decreased (p<0.001) from baseline during LBNP with no effect of FLZ (PLA: -31.8 ± 19.4 vs. FLZ: -36.1 ± 25.1 p:0.53, d: 0.17). Interestingly, FVC increased from baseline to exercise (p<0.001) with no effect of FLZ (PLA: 819.6 ± 503.9 vs. FLZ: 709.1 ± 459.2 p: 0.36, d: 0.20). However, FVC was unaltered during Ex+LBNP in PLA from steady-state exercise (4.5 ± 13.9, p:0.32) but was significantly reduced with FLZ (–11.5 ± 13.3, p>0.001) with a large effect size between conditions (p>0.001, d: 1.1). All responses were similar between the sexes (p:0.5, d:0.3). CONCLUSION: Our results indicate that in young healthy adults, inhibition of cytochrome P450 pathways attenuated vascular opposition to superimposed sympathetic stress during dynamic handgrip exercise. Thus, cytochrome P450 pathways are mechanistically involved in functional sympatholysis in young healthy humans.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Bacterial lipopolysaccharide (LPS) inhibits colonic uptake of the microbiota-generated forms of vitamin B1 [thiamin pyrophosphate (TPP), and free thiamin] via different mechanisms
Authors: Anthonymuthu Selvaraj , Sabui Subrata , Lee Katherine , Sheikh Alaullah , Fleckenstei James , Said Hamid
Published: May 2023

LPS is a major component of the outer membrane of Gram-negative bacteria. The gut is exposed to high levels of this endotoxin in conditions such as IBD, necrotizing enterocolitis, and sepsis. LPS affects different aspects of cell physiology including transport at the cell membrane. Effects on the latter, however, are differential; and both induction and inhibition in substrate uptake have been observed. Nothing is currently known about the effect of LPS on colonic uptake of the microbiota-generated forms of vitamin B1, i.e., TPP and free thiamine. We investigated this issue in the current study using human-derived colonic epithelial NCM460 cells, human native differentiated and polarized colonoid monolayers, and mouse intact colonic tissue preparation together with appropriate cellular and molecular approaches. The results showed that exposure of colonic epithelial cells to LPS leads to a significant inhibition in carrier-mediated [3H]-TPP uptake. This inhibition was associated with: (i) a significant reduction in level of expression of the colonic TPPT transporter (cTPPT; SLC44A4) protein and mRNA; (ii) a significant inhibition in SLC44A4 promoter activity (associated with suppression in level of expression of the transcription factor Elf-3, which is need for promoter activity); the inhibition also appeared to be mediated via NF-κB, p38 and JNK (MAPK) mediated pathways. Similarly, LPS treatment of colonic epithelial cells was found to lead to a significant inhibition in colonic carrier-mediated uptake of free [3H]-thiamin. This inhibition, however, was not associated with changes in level of protein and mRNA expression of the involved transporters, i.e., THTR-1 & -2. These results demonstrate for the first time that exposure of colonic epithelial to LPS leads to significant inhibition in colonic uptake of the microbiota-generated TPP as well as free thiamin. The inhibition, however, appears to be mediated via different cellular mechanisms.

This study was supported by NIH grant DK-56061, and VA grants 101BX001142 & IK6BX006189 to HMS; NIH grant AI089894 and AI126887 and VA grant 5I01BX001469-05 to JMF.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Female Mice Are Less Susceptible to the Effects of Diet-Induced Obesity on Sleep and Sleep-Disordered Breathing
Authors: Kim Lenise , Pho Huy , Pham Luu , Polotsky Vsevolod
Published: May 2023

Diet-induced obesity (DIO) in male mice is a well-established model of sleep-disordered breathing (SDB). Male DIO mice manifest obesity hypoventilation, increased apnea severity, and sleep fragmentation. The effects of DIO on breathing during sleep and sleep architecture in females have been insufficiently investigated. In this study, we aimed to compare the breathing and sleep patterns of lean and obese female mice of similar genetic background. We hypothesized that female mice are less susceptible to the detrimental effects of DIO on sleep and SDB compared to males. Full-polysomnographies were performed in 15 lean female C57BL/6J and 14 female DIO-C57BL/6J mice using a whole-body plethysmography chamber. Arousals from sleep and apneas were manually scored. Ventilatory response to an arousal was calculated as ratio of the peak minute ventilation (VE) during each arousal relative to the baseline VE (from 2.5s to 5s preceding the arousal). Breathing stability was measured using Poincaré plots of VE. Mice were acutely exposed to 8% CO2 to measure the hypercapnic ventilatory response (HCVR). An additional 10 mice (5 each/group) underwent 24-h metabolic studies. We found that obesity was associated with hypometabolism, indicated by reduced oxygen consumption (VO2) and CO2 production in DIO mice. VE in 8% CO2 and HCVR were significantly suppressed suggesting a decreased CO2 sensitivity in obese animals. NREM sleep duration was reduced, but REM sleep was preserved. Arousal frequency was similar between groups (~78 events/h). DIO mice hypoventilated during NREM sleep. However, apnea severity was 4-fold augmented in lean females compared to DIO mice (14.7±3.9 vs 74.2±11.9 events/h; P<0.001). Breathing stability during sleep was significantly increased in lean mice. Ventilatory response to an arousal was higher in lean females, but it was attributed to an overall increased VE during arousal and at baseline. Our results suggest potential sex differences in the effects of DIO on breathing during sleep and sleep architecture. DIO in females appears to be less detrimental regarding SDB severity. The mechanisms underlying the augmented SDB severity in lean females are unknown, but an exacerbated CO2 responsiveness during sleep could play an important role.

American Heart Association Postdoctoral Fellowship Award 828142 (to L.J.K.), NHLBI grant NIH R01 HL128970, R01 HL133100, and R01 HL13892 (to V.Y.P.), American Academy of Sleep Medicine Foundation 238-BS-20, and American Thoracic Society Unrestricted Award (to L.V.P.)

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Longitudinal study of renal phenotype in mouse model of sickle cell trait
Authors: Sako Mohammad , Key Nigel , Pawlinski Rafal , Kasztan Malgorzata
Published: May 2023

Background: Sickle cell disease (SCD) is an autosomal recessive blood disorder caused by mutations in the β-globin gene, resulting in hemoglobin S (HbS) polymerization and red blood sickling. SCD patients carry two copies of the mutant HbS (HbSS) and develop anemia, iron overload and to end-organ damage. Heterozygous carriers (AS) are said to have sickle cell trait (SCT). While not associated with shortened life expectancy, individuals with SCT are at increased risk for a limited number of complications, including chronic kidney disease (CKD) that has yet to be fully elucidated. Our objective was to examine if mouse model of SCT is associated with worsening kidney function over time, mimicking an increased risk of CKD in SCT. Methods: We used male humanized sickle cell trait (HbAS) and age-matched genetic control (HbAA) mice (n=18). Analysis of urinary renal biomarkers (albuminuria, proteinuria, kidney injury marker 1 (KIM-1), osmolality) and time course of kidney function (determined via transcutaneous measurement of glomerular filtration rate (GFR)) were conducted in 4-week intervals from 12 to 40 weeks of age. Results: At 8 weeks of age, HbAS mice exhibited no differences in GFR, or proteinuria compared to HbAA controls. Significant increase in GFR, but not proteinuria, was observed in HbAS at 16 weeks of age compared to HbAA mice (241.6±5.3 ul/min vs 218.2±5 ul/min, respectively, p ˂0.05). Importantly, significant loss of kidney function measured by GFR (200 ± 3 vs 217.9 ± 3.2 ul/min, p<0.05) and twofold increase in proteinuria (4.6 ±1.1 vs 2.3 ±0.4 mg/24h, p<0.05) were observed at 36 weeks old HbAS mice when compared to age-matched controls. Following the increase in GFR at 16 weeks of age, HbAS developed mild degree of glomerular damage presented with subsequent slow increase in albuminuria over time (pgen<0.0001; page<0.0001, pinter=0.001). Interestingly, increased levels of urinary KIM-1 excretion, marker of tubular damage, were observed in HbAS mice throughout the duration of the study, with the highest levels at 16 weeks (228.3 ± 32.6 pg/24h vs 53.8 ± 8.5 pg/24h, respectively, p˂0.05). Urine osmolality was not different at 8 weeks of age, but progressively declined throughout the duration of the study in HbAS mice vs controls (1946 ±63 vs 2672 ±135 mOsm/kg at 36 weeks, p=0.002). Conclusion: Humanized sickle cell trait mice develop progressive loss of kidney function and associated renal injury over time.

Funding: NHLBI–R00HL144817 and NHLBI-R01 HL157441

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Bmal1 contributes to mean blood pressure rhythms in rats on a low salt diet
Authors: Monroe Brianna , Guerrero Samuel , Rhoads Megan , Pollock David
Published: May 2023

Bmal1 is a core component of the molecular clock which is responsible for generating circadian rhythms in many physiological parameters, including blood pressure (BP). Sodium intake can affect circadian rhythms, as high salt consumption can increase BP and alter the expression of the molecular clock. However, the effect of low salt consumption on the circadian rhythm of BP and hormones crucial to sodium homeostasis has not been extensively studied. Using a whole-body Bmal1-knockout (KO) rat, we wanted to determine the contribution of the molecular clock to circadian BP rhythms during low salt diet (LSD, 0.0049%).We hypothesized that Bmal1-KO rats on a LSD would have lower mean BP and higher aldosterone levels in comparison to littermate controls on LSD. Male and female Bmal1-KO rats and littermate controls (WT) had telemeters implanted in the abdominal aorta at 8-10 weeks old. After a 10-day surgical recovery period, BP was continuously recorded while rats were maintained on ad libitum normal salt diet (0.49% NaCl, NSD) for 1 week. Rats were placed in metabolic cages and 12-hour urine samples collected at ZT0 and ZT12 for two days. Next, rats were placed on LSD for one week while maintaining BP measurements. This was again followed by two day, 12-hour urine collections. BP was averaged hourly and analyzed for circadian rhythmicity (cosinor). The MESOR for mean arterial pressure was significantly higher in WT (n=10, 4/10 male) and KO groups (n=8, 4/8 male) on NSD (WT: 108±2 mmHg; KO: 104±2 mmHg) compared to LSD (WT: 104±2 mmHg; KO: 102±2 mmHg; p<0.05 for diet, 2-way ANOVA). BP amplitude was significantly higher in WT rats (NSD: 7.6±0.6 mmHg; LSD: 6.9±0.7 mmHg) compared to KO rats (NSD: 5.2±0.4 mmHg; LSD: 5.3±0.9 mmHg) (p<0.05 for genotype). BP acrophase was similar between groups on both diets. Urinary aldosterone excretion (ELISA) was similar between WT and KO rats on NSD (Daytime: WT 26±5 pg/12hrs, KO 16±1 pg/12hrs; Nighttime: WT 48±8 pg/12hrs, KO 46±4 pg/12hrs) and on LSD (Daytime: WT 95±38 pg/12hrs, KO 109±44 pg/12hrs; Nighttime: WT 174±63 pg/12hrs, KO 155±47 pg/12hrs), with statistically significant overall effects of diet and time of day (p<0.05, 3-way ANOVA). Together, these data support a role for Bmal1 in control of BP amplitude in rats on NSD and LSD. This does not appear to involve changes in aldosterone since urinary excretion followed predicted patterns by time of day and salt diet, but were not impacted by loss of Bmal1 in ad libitum fed rats.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Brachial artery flow-mediated dilation during incremental handgrip exercise is not impacted by sex or female sex hormones
Authors: Annis Meghan , D'Souza Andrew , Coombs Geoff , Manabe Kazumasa , Sanchez Belinda , Fu Qi , Shoemaker Kevin
Published: May 2023

Sex differences exist in reactive hyperemia flow-mediated dilation (RH-FMD), with males demonstrating larger RH-FMD responses than naturally menstruating females and females using oral contraception (OC). This difference is thought to be partly attributed to sex hormones. Among females, some studies demonstrate lower RH-FMD in females using OC relative to naturally menstruating females. To date, non-invasive assessments of endothelial function in males, naturally cycling females and females using OC have only been completed using RH-FMD. Notably, due to differences in the shear stimulus during sustained shear-induced FMD (SS-FMD) versus the transient pattern observed with RH-FMD, SS-FMD may provide distinct insight into endothelial dysfunction undetected with RH-FMD. Therefore, we hypothesized that the SS-FMD responses to incremental handgrip (IHG) exercise would be greatest in males, and that females using OC would have the smallest increase in SS-FMD compared to males and naturally menstruating females. The IHG protocol consisted of 3-minute stages of rhythmic handgrip exercise at 15, 30, and 45% of an individual’s maximal voluntary contraction (MVC) force, with no rest period between MVC transitions. Brachial artery diameter and blood velocity were measured simultaneously via duplex Doppler ultrasound in 10 males (26±4 [mean±SD] years), 11 naturally menstruating females (25±4 years), and 9 females using OC (27±5 years) during IHG. Females were tested in the mid-luteal (ML) and active (high hormone) OC phases. SS-FMD and shear rate were analyzed using linear mixed model analyses. MVC was greater in males relative to both groups of females (males: 40±6, ML: 28±8, OC: 26±6 kg; One-way ANOVA: post-hocs P≤0.001). Brachial artery shear rate was not different between males, ML females or females using OC throughout exercise (group-by-stage interaction: P=0.975). Consequently, the brachial artery dilated by 1.3±1.5, 0.1±2.1, and 1.2±3.6 % at 15% MVC, 4.0±3.1, 2.7±3.3, and 4.1±4.8 % at 30% MVC, and 9.1±4.9, 7.3±5.4, and 7.6±5.9 % at 45% MVC, in males, ML females, and females using OC, respectively (all P>0.05). Furthermore, the slopes of the relationship between the change in brachial artery diameter and shear rate was similar between the three groups (Males: 0.0004±0.0002, ML: 0.0003±0.0002, OC: 0.0003±0.0002 Δmm/Δ1·s-1; One-way ANOVA: P=0.564). Taken together, these data indicate that endothelial function in response to sustained elevations in shear stress are not impacted by biological sex or OC use.

Supported by the Natural Sciences and Engineering Research Council of Canada, and IEEM indirect funds.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Interferon regulatory factor 4 dictates metabolic flexibility of fat versus muscle loss during repeated intermittent fasting
Authors: Marko Daniel , McAlpin Breanne , Duggan Brittany , Cavallari Joseph , Schertzer Jonathan
Published: May 2023

Interferon regulatory factor 4 (IRF4) is a transcription factor that regulates the proliferation, differentiation, and metabolism of immune cells, such as macrophages and T cells. Recently, IRF4 has been shown to regulate lipolysis in adipocytes and increases in visceral human adipose tissue and murine white adipose tissue during acute fasting. However, it was unknown how IRF4 influences metabolic responses to repeated intermittent fasting. We hypothesized that blunted lipolysis, caused by Irf4 deletion, would promote altered metabolic substrate selection, specifically amino acids from muscle as opposed to fatty acids and glycerol from fat, to maintain energy homeostasis during repeated fasting in mice. Littermate whole body and adipocyte specific Irf4 knockout mice were placed on a 45% kcal from fat diet and either left to consume food ad-libitum or put on a 5:2 repeated fasting protocol for 10 weeks, which consisted of two 24-hour fast periods on non-consecutive days of the week. Body composition measures, using MRI, showed that littermate wild type mice lost more body and fat mass compared to Irf4 knockout mice during the fasting protocol. In line with our hypothesis, Irf4 knockout mice lost significantly more lean mass compared to wild-type mice. In conjunction with our MRI data, the Irf4 knockout mice had lower hindlimb, tibialis anterior muscle mass along with higher gonadal and inguinal white adipose tissue mass after the fasting protocol compared to the wild type mice. Additionally, we found that adipocyte-specific deletion of Irf4 recapitulated these effects on muscle and fat mass during intermittent fasting. We then used metabolic cages to examine energy expenditure and fuel oxidation. Complementary to our previous data, adipocyte-specific Irf4 knockout mice had lower fatty acid oxidation and higher carbohydrate oxidation rates compared to the wild type mice. Since adipocyte-specific Irf4 knockout mice have blunted lipolysis and lower fatty acid oxidation rates, our data suggests that another metabolic fuel source, specifically muscle protein, is being used to maintain energy homeostasis during fasting. Collectively, our data highlights a novel metabolic role for IRF4 in mediating fasting-dependent changes in body composition, specifically the balance between fat and muscle mass during repeated intermittent fasting.

CIHR, NSERC, CFI, and Diabetes Canada

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Western diet exacerbates aristolochic acid-induced nephropathy in mice
Authors: Oe Yuji , Kim Young Chul , Kanoo Sadhana , A Goodluck Helen , Lopez Natalia , Maher Pamela , Vallon Volker
Published: May 2023

Aristolochic acid (AA) ingestion causes Balkan nephropathy, characterized by tubular injury and progression to chronic kidney disease (CKD). AA is taken up by proximal tubule cells via organic anion transport (OAT3) and induces p53-mediated DNA damage, but little is known about modulating factors. The Western diet (WD) is rich in saturated fats, sugars, and salt and contributes to the development of metabolic disorders such as obesity and diabetes, but also to the progression of CKD. The aim of this study was to elucidate the impact of WD on AA-induced kidney injury. 5-week-old male C57BL6 mice were fed with WD or normal chow (NC) for 8 weeks, followed by administration of AA every 3 days at a dose of 3 mg/kg for 3 weeks. Samples were collected after a 3-week recovery period (n=4/group for vehicle; n=7-8/group for AA). The AA-induced increase in plasma creatinine and the reduction of hematocrit were significantly greater in WD vs NC. This was associated with increased kidney gene expression in WD vs NC of markers of DNA damage ( p53), injury ( Kim1 and Ngal), and inflammation ( Tnfa). The WD group had a greater body weight and, therefore, received a higher total dose of AA (0.73 and 0.6 mg), which may have worsened kidney injury. Therefore, in a second series, the same total dose of AA (0.9 mg) was administered to WD and NC groups. WD similarly increased the AA-induced fall in hematocrit and rise in plasma creatinine and renal p53, Kim1, Ngal, and Tnfa mRNA expression compared to the NC group. Moreover, WD increased renal mRNA expression of Oat3. These findings suggest that WD increases the susceptibility to AA nephrotoxicity, possibly through upregulation of OAT3. This model may be useful for studying the mechanistic impact of Western diet on the development and progression of CKD caused by nephrotoxic agents.

Supported by National Institutes of Health (NIH) Grants RF1AG061296 (PM, VV), R01DK132690 (VV), and P30 DK079337 (VV), the Department of Veterans Affairs, and a fellowship of the Manpei Suzuki Diabetes Foundation (YO).

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Intermittent activation of large diameter phrenic afferents induces phrenic motor plasticity
Authors: Popp Nicole , Holmes Taylor , Streeter Kristi
Published: May 2023

We recently showed that in anesthetized rats, 30 minutes of electrical stimulation applied to the diaphragm muscle induces phrenic afferent dependent plasticity characterized by a long-lasting increase in diaphragm electromyography amplitude. Our previous data also indicate that large diameter phrenic afferents may be most effective at eliciting plasticity. Here we directly test the hypothesis that intermittent activation of large diameter phrenic afferents induces phrenic motor plasticity. In urethane anesthetized and mechanically ventilated male Sprague-Dawley rats, we isolated the left and right phrenic nerves. We applied inspiratory-triggered electrical stimulation (biphasic; 40Hz; 160mA) to one phrenic nerve in a sustained (n= 5, 25 minutes) or intermittent (n=5; 5 X 5-minute episodes) pattern, and recorded contralateral motor output before, during, and for 60 minutes after stimulation. We used a narrow pulse width (0.1ms) to preferentially activate large diameter afferents. At 60 minutes, rats receiving sustained stimulation showed no difference in contralateral phrenic motor amplitude compared to time controls not receiving stimulation (n=5; P=0.1320). In contrast, rats receiving intermittent stimulation had an increase at 60 minutes compared to time controls (P=0.0005) and sustained stimulation (P=0.0024). This suggests that intermittent, but not sustained, phrenic nerve stimulation induces contralateral phrenic motor plasticity. To test the hypothesis that phrenic afferents are necessary for intermittent induced plasticity, separate animals received either a left, C3-C6 dorsal rhizotomy (n=3) or laminectomy (n=4) prior to intermittent stimulation. Laminectomy controls expressed a long-lasting increase in contralateral phrenic motor amplitude compared to time controls (n=5) at 60 minutes after stimulation (P=0.0112). Animals receiving a dorsal rhizotomy were not different than time controls at 60 minutes (P=0.4058), indicating phrenic afferents are necessary for the induction of plasticity. These data show for the first time that intermittent activation of large diameter phrenic afferents induces phrenic motor plasticity. A detailed understanding of afferent mediated plasticity will be beneficial in the development of therapeutic techniques utilizing electrical stimulation of the diaphragm muscle (i.e., diaphragm pacing).

This work was supported by funding from the National Institute of Health, grant numbers: R00 HL143207-01 (KAS). The authors declare no competing financial interests.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Students prefer final projects that are scaffolded to avoid procrastination and negative impact on mental health
Authors: Lopez Savannah , Hsu Jeremy , Halpin Patricia
Published: May 2023

Many life science classes include final projects. Such projects are often due at the end of the term, though may involve several sub-parts that students can work on throughout the term. Instructors can take different approaches to these sub-parts, with some instructors requiring student submission throughout the term and others only collecting the project at the end. Our project aimed to investigate if students prefer having instructors scaffold a final project, and their reasons behind their preferences. Methods- We surveyed students from a range of colleges from community college through professional school. By using a Qualtrics survey that was sent to their instructors for disbursement we were able to collect 324 responses to both closed- and open-ended questions. For open-ended questions, each researcher independently generated a codebook using 18% of samples, before the research team discussed to generate a consensus codebook. Interrater reliability was checked with another 18% of the responses. Fleiss’s kappa was 0.65, indicating substantial agreement. Thus, one researcher coded the remaining responses. Results- The majority of students with final projects (69.45%) stated that they preferred to have their final projects broken into parts with separate due dates. Students primarily stated that they preferred this scaffolding because it keeps them on track, reduces procrastination and makes the project more manageable (55.12% of respondents). The second most common response provided (15.12 %) was that students preferred having the project broken into smaller parts led to less stress or anxiety, decreased feeling of being overwhelmed and helped reduce stress at the end of the term. Of those that stated they did not prefer to have the project scaffolded the primary reason was that scaffolding caused more pressure, anxiety, stress or changed their perception of the project. In conclusion using scaffolding to design and assign final projects benefits the student because they can work on separate parts of the assignment throughout the term and not wait until the end of the semester to begin. Using this project design can also reduce negative impacts on mental health. The results of this study can inform instructors of a way to design final projects that minimize student procrastination while decreasing negative impacts on mental health and aids in student management of the assignment

Funded by Promoting Active Learning and Mentoring (PALM) Fronds grant.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Sex differences in oxidative stress-mediated reductions in microvascular endothelial function in young e-cigarette users
Authors: Halstead Kristen , Wetzel Elizabeth , Brustkern Kaila , Stanhewicz Anna
Published: May 2023

Tobacco and nicotine use cause vascular endothelial dysfunction, increasing lifetime cardiovascular disease risk. Chronic e-cigarette use is reported to decrease endothelial function, however the mechanism(s) mediating this reduction remain unclear. Using the cutaneous circulation as a model, we examined endothelium- and nitric oxide (NO)-dependent dilation, and the role of oxidative stress in attenuating these responses, in otherwise healthy, young chronic (≥6 months) e-cigarette users (EC) compared to healthy controls (HC). We hypothesized that: EC would have reduced endothelium- and NO-dependent dilation, and local tempol (superoxide scavenger) administration would increase these responses in EC. We further examined the effect of sex within HC and EC and hypothesized that female EC would have the greatest reduction in endothelium- and NO-dependent dilation. 20 HC (21±2 yrs; 10M/10F) and 18 EC (21±2 yrs; 10M/8F) underwent a standard local heating protocol (42°C; 0.1°C·s-1). Two intradermal microdialysis fibers were placed in the ventral forearm skin for local delivery of lactated Ringer’s (control), or 10μM tempol. After full expression of the local heating response, 15mM NG-nitro-L-arginine methyl ester (L-NAME; NO synthase-inhibition) was perfused. Red cell flux was measured continuously by laser-Doppler flowmetry, and cutaneous vascular conductance (CVC=flux/MAP) was standardized to maximum (%CVCmax; 28mM SNP + 43°C). Between groups, endothelium- (EC: 73 ± 15 vs HC: 87 ± 9 %CVCmax; p<0.001) and NO-dependent (EC: 48.1± 17.0% vs. HC: 61.8 ± 14.7%; p=0.011) dilation were attenuated in EC compared to HC. Local tempol perfusion increased endothelium- (83.8 ±12.1% p=0.007) and NO-dependent (61.4 ± 14.2% p=0.011) dilation in EC but had no effect in HC (all p>0.05). Within sex, female EC had lower endothelium- (EC: 69.9±3.4 vs HC: 89.1±3.5 %CVCmax; p<0.001) and NO-dependent (EC: 46.9±5.5 vs HC: 69.4±5.0%; p=0.005) dilation compared to female HC, but there were no differences between these responses in HC and EC males (both p>0.05). Tempol perfusion augmented endothelium- (83.4±4.4 %CVCmax; p=0.31) and NO-dependent (60.2±5.1%; p=0.04[AS1] ) dilation in EC females but had no effect in EC males (both p>0.05). These data suggest that otherwise healthy young adult e-cigarette users have reduced microvascular endothelium- and NO-dependent dilation. Within e-cigarette users, these results are driven by greater reductions in females, and superoxide contributes to these impairments.

Supported by NIEHS/NIH P30 ES005605

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Cross talk between histone 3 acetylation, methylation and phosphorylation by intermittent hypoxia.
Authors: Nanduri Jayasri , Wang Ning , Prabhakar Nanduri
Published: May 2023

We have previously identified histone 3 (H3) post translational modifications, specifically acetylation and methylation as an early epigenetic mechanism associated with sympathetic nerve activation and hypertension in rodent models of intermittent hypoxia (IH), a hallmark feature of sleep apnea (Nanduri et al, Physiol genomics 2021, Ning et al, Front Physiol 2021). Phosphorylation of H3 serine (S) residues that are adjacent to acetylation and methylation sites are also shown to regulate chromatin structure and gene expression. The aim of the present study is to assess whether IH affect H3 phosphorylation and whether phosphorylation influences H3 acetylation by IH. Studies were performed on rat pheochromocytoma (PC12) cells and adrenal chromaffin cells of rats treated with 10d IH as well as heme oxygenase (HO)-2 null mice, which exhibit spontaneous apneas. IH increased H3 lysine (K) 27 acetylation in PC12 cells, rat adrenal chromaffin cells and HO-2 null mice. These effects were associated with dephsophorylation of H3 at S28 and demethylation of K27. Inhibition of S28 dephosphorylation by protein phosphatase inhibitor (PIC) or Calyculin A, a specific inhibitor of protein phosphatase (PP)-1 and -2, inhibited H3K27 acetylation by IH. Conversely, inhibition of K27 acetylation with CTK7A a specific inhibitor of acetyl transferase p300/CBP increased S28 phosphorylation and K27 methylation in IH exposed cells. These results demonstrate that effects of IH on H3 lysine acetylation, methylation and phosphorylation are complex and a cross-talk between the three events regulate IH-induced transcriptional activity.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Early Interleukin-6 Mediated Activation of SGK1 is Primarily via Variant 6
Authors: Nnah Favour , Yoshigi Masaaki , Wynne Brandi
Published: May 2023

Hypertension is associated with inflammation, including elevated interleukin-6 (IL-6) levels. IL-6 is an inflammatory cytokine, which we have previously shown to activate the mineralocorticoid receptor (MR) and the downstream sodium (Na) transporters- Na-chloride cotransporter (NCC) and the epithelial Na channel (ENaC). However, how IL-6 activates the MR is still unknown. IL-6 is also linked to Serum and Glucocorticoid-regulated Kinase 1 (SGK1), which is a downstream effector of the MR. SGK1 regulates both NCC and ENaC, increasing activity and tubular reabsorption of Na. This increased Na reabsorption favors water retention, thus increasing blood pressure. Previously, IL-6 has also been linked with SGK1 activation. We hypothesize that IL-6 increases SGK1 mRNA and protein expression in renal epithelial cells. To test this, we used in vitro renal epithelial cell models of both the late distal convoluted tubule (DCT2) and inner medullary collecting duct (IMCD), mDCT15 and IMCD3, respectively. Cells were treated with an IL-6 time-course (100ng/ml for 1, 3 and 6 hours). There are 6 known transcriptional variants for SGK1. We designed variant-specific PCR primers and, a pilot study based on regular PCR concluded that expression levels of only variants 5 and 6 (V5, V6) are significant. Using Real Time quantitative RT-PCR, we were then able to quantify transcript levels of both SGK1 V5 and V6 variants. With IL-6 treatment, we observed a 160%-211% increase at early time points (30mins-1hr) in SKG1 V6 and a lesser response (26%-88% increase) with V5 as compared to vehicle treated mDCT15 cells. Since SGK1 has been observed in the membrane fraction and in the cytoplasm, protein extracts from cells were fractionated (mPER kit) to assess membrane and cytosolic SGK1 protein levels using immunoblots. Densitometric analysis of immunoblots demonstrated a 5.5- and 3.1-fold increase in SGK1 protein expression at 1hr in mDCT15 and IMCD3 cells, respectively. These data suggest that IL-6 induces increases in SGK1 transcription and translation. Interestingly, we also found that majority of SGK1 was in the membrane fraction and the level was consistent. Together, these data suggest that SGK1 V6 may be responsible for the increased sodium reabsorption in response to IL-6 stimulation in renal epithelial cells.

NIH funding

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Social isolation stress favors cardiometabolic comorbidities of sleep apnea
Authors: Gagnon Marianne , Fournier Stéphanie , Marcouiller François , J. T. Arnold Alexandre , Joseph Vincent , Michael Natalie , Kinkead Richard
Published: May 2023

Sleep apnea (SA) is a multifactorial and sex-specific respiratory disorder characterized by repeated respiratory arrests during sleep. However, the origins of this disease are not clearly identified. Chronic stress increases risk for obesity, hypertension, and depression, which are common comorbidities of SA. With the increasing prevalence of obesity and about one-third of the world population reported feeling stressed, the study of the impact of stress on respiratory homeostasis has never been so relevant. Since chronic stress induces the main comorbidities of SA, we hypothesized that social isolation (SI) stress induces sex-specific cardiorespiratory and metabolic disorders in rats similar to patients with SA.80 Sprague Dawley male and female rats (8 weeks old) were kept in: 1) standard housing (2/cage; controls) or in 2) social isolation (1/cage). At the end of the three-week protocol, apneas were quantified by whole-body plethysmography during sleep; respiratory variability was assessed by Poincaré plot analysis. Cardiovascular measurements were obtained by tail cuff, body composition by preclinical magnetic resonance, and plasma leptin and corticosterone levels by multiplex immunoassay. The amount of food was weighed before and after the experiments to calculate the intake.By comparison with controls, SI elevated corticosterone levels in male but not females. SI increased the apnea index in intact rat by 21% and augmented respiratory instability. SI also increased blood pressure (+11mmHg) and food intake (+11g/day); however, females had lower increases than males. In males, SI increases weight gains, fat mass, and decreases plasma leptin levels; these effects were not observed in females.SI promotes the emergence of apnea and respiratory instability during sleep. Since the cardiorespiratory and metabolic disturbances are similar to clinical features of SA patient, we propose that stress plays an important role in the etiology of SA. SI offers a novel and unique opportunity to study the origins and pathophysiology of SA.

This research is supported by operating grants from the Canadian Institutes of Health Research (CIRH, RK and VJ). N.J.M. was supported by a Sentinel North Partnered Research Chair in Sleep Pharmacometabolism and a Fonds de Recherche du Quebec Sante (FRQS) Research Scholar J1 award. MG is the recipient of a doctoral scholarship from the FRQS.

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
Senolytics do not prevent muscle loss in Sod1 knockout mice
Authors: Borowik Agnieszka , Lawrence Marcus , Piekarz Katarzyna , Miller Benjamin , Van Remmen Holly , Brown Jacob
Published: May 2023

The accumulation of senescent cells is a hallmark of aging and age-related disease. Cellular senescence is a state of cell cycle arrest resulting, in part, from metabolic stress and reactive oxygen species (ROS) production. The importance of senescent cells in sarcopenia is underexplored with limited data for or against their contribution to the age-related loss of muscle mass and function. Oxidative stress is an imbalance between ROS production and neutralization. Mice with a knock-out of the endogenous antioxidant superoxide dismutase 1 (Sod1KO) have high levels of oxidative stress and a phenotype resembling accelerated frailty. Because of this association of oxidative stress and cellular senescence, we reasoned that Sod1KO mice would have a high senescence burden. Recently, the senolytic treatment of dasatinib and quercertin (DQ) in Sod1KO mice eliminated senescent cells and restored markers of liver health. Therefore, we hypothesized that reduction of senescent cells with DQ could prevent sarcopenia in Sod1KO mice. To test this hypothesis, we treated wildtype (WT) and Sod1KO mice with DQ or vehicle (Veh) from 6 months of age, the age at which the Sod1KO mice showed first signs of sarcopenia. Sod1KO mice had reduced muscle mass compared to WT mice, however, there was no effect of DQ treatment. In contrast, after 7 months of DQ treatment, the specific force generated by the extensor digitorum longus muscle in Sod1KO mice was greater than untreated by ~11% (Sod1KO-Veh 14.542 ± 0.550 N/cm2; Sod1KO-DQ 16.212 ± 1.464 N/cm2, p=0.013), although there were no differences between Veh and DQ in maximum force. There was a main effect of genotype in fragmentation of the neuromuscular junction in Sod1KO, regardless of DQ treatment (WT-Veh 0.87 ± 1.52% vs Sod1KO-Veh 77.10 ± 2.09&%, p <0.001; WT-DQ 0.0 ± 0.0% vs Sod1KO-DQ 70.5 ± 18.1%, p <0.001). We also observed a genotype effect in protein turnover analysis, as Sod1KO mice had higher mitochondrial and myofibrillar protein synthesis rates than WT mice, while there was no effect of DQ treatment. There was a trend for greater maximally stimulated complex I respiration in muscle mitochondria from Sod1KO-DQ mice compared to Sod1KO-Veh mice (97.32 ± 29.47 vs 61.78 ± 33.89 pmol/(s·mg), p=0.065, respectively). However, there were no effects of genotype or treatment in maximally stimulated complex I+II and complex II respiration, as well as hydroperoxide generation among groups. Our study suggests that DQ senolytic treatment does not prevent oxidative stress-induced sarcopenia in Sod1KO mice.

P01AG051442, 1 IK6 BX005234, 1 IK2 BX005620-01A1

This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.