bluuebunny/crossref_metadata_2025 huggingface dataset:
Bone marrow macrophage migration is dependent on ASIC2 and betaENaC
Authors: Drummond Heather , Pittman Katarina , Wasson Robert , Fleming Adam , Granger Joey
Published: May 2023

Monocyte-macrophages play an important role in phagocytosis of pathogens and cellular debris following infection or tissue injury in several pathophysiological conditions. Naïve macrophages (Mf), can be polarized into proinflammatory (M1), and/or anti-inflammatory (M2) phenotypes. M1 cells are more phagocytic and adhesive/less motile than anti-inflammatory M2 macrophages. A previous study from our laboratory in the RAW monocyte-macrophage cell line suggests that polarization to M1 macrophages involves inhibition of Epithelial Na+ Channel (ENaC) expression, leading to loss of motility. To determine if a similar relationship between ENaC and migration exists in bone marrow macrophages (BMM), we examined chemotactic migration in BMMs from mice lacking βENaC and ASIC2a (KO). BMMs were harvested from the femurs of male and female WT and KO mice (6-12 weeks of age), expanded in culture with colony stimulating factor 1 (CSF1, 20 ng/ml) and cryopreserved for later use. Chemotactic migration responses were inhibited in BMM from KO male (94±4 vs 60±2 cells/FOV, n=35-42 FOVs) and female (142±5 vs 74±3 cells/FOV, n=42) mice. ENaC blockade using 1 μM amiloride inhibited migration in WT BMMs equivalent to migration in untreated KO BMMs and had no further inhibitory effect on KO BMM migration. These findings suggest βENaC and/or ASIC2 account for up to 50% of chemotactic migration in BMM and 100% of amiloride-dependent migration. BMM migration responses were rescued in stable transfected male KO BMMs with βENaC (88±4) or ASIC2a (77±5) compared to the EGFP_control KO (60±2 cells/FOV), providing further evidence of the importance of βENaC and/or ASIC2 in BMM migration. Polarization of male BMMs with IFNg (10 ng/mL, 18 hr) inhibited migration in BMM from WT (64±3 vs 22±1 cells/FOV) and KO (48±2 vs 16±1) animals, suggesting KO BMMs do not behave like fully polarized M1 macrophages. Taken together, our findings indicate that βENaC and ASIC2a play an important role in BMM migration response. Loss of βENaC/ASIC2a leads to a low migratory phenotype like M1 macrophages. Restoration of βENaC or ASIC2 rescues BMM migration. Targeting ENaC activity/expression in BMMs may limit their ability to migrate to sites of injury. Whether this is benefical or detrimental to non-infectious tissue injury is unclear.

NIH R01HL136684, P20GM104357, and P20GM121334.

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.
Insulin-Derived Endothelial Microvesicles Induce a Proinflammatory Endothelial Phenotype
Authors: Ostrander Emily , Garcia Vinicius , Fandl Hannah , Levy Ma'ayan , Stockelman Kelly , Goulding Anabel , DeSouza Noah , Greiner Jared , Hijmans Jamie , DeSouza Christopher
Published: May 2023

Increased inflammation renders the vascular endothelium susceptible to atherosclerosis and thrombosis. Insulin has been shown to influence vascular function. Hyperinsulinemia is associated with a proinflammatory endothelial phenotype. In vitro, insulin has been shown to increase the inflammatory transcription factor NF-kB. Extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), have emerged as mechanistic factors mediating of endothelial inflammation and, in turn, vascular disease. Circulating EMVs are elevated in metabolic conditions associated with hyperinsulinemia. However, whether insulin stimulates EMV release is unknown, and, if so, whether insulin-derived EMVs are proinflammatory. The experimental aims of this study were to determine: 1) if insulin stimulates EMV release from endothelial cells; and 2) whether insulin-derived EMVs promote a proinflammatory endothelial phenotype. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and incubated with 1 nM insulin (concentration representing a hyperinsulinemic state) for 24 h. EMVs released into the supernatant from cells treated with insulin or vehicle were isolated and quantified by flow cytometry (CD144-PE). In separate experiments, HUVECs (2x106 cells/condition) were treated with either insulin, insulin-induced EMVs or control EMVs for 24 h. EMV release was markedly higher (~185%; P<0.05) in cells treated with insulin compared with control (88±10 vs. 31±5 EMV/μL). Insulin-generated EMVs induced significantly higher release of interleukin (IL)-6 (25.4±1.5 vs 20.1±1.2 pg/mL) and IL-8 (37.8±2.5 vs 27.1±2.7 pg/mL) than control EMVs. Although total NF-kB p65 expression was not significantly altered; insulin-derived EMVs induced ~30% higher (P<0.05) expression of phosphorylated-NF-kB p65 (Ser536; active NF-kB) (8.5±0.4 vs. 6.5±0.3 AU). The adverse effects of insulin-generated EMVs on IL-6, IL-8, and pNF-kB p65 were consistent with the proinflammatory effects of insulin. In summary, insulin induces an EMV phenotype that adversely affects endothelial cell inflammation. Insulin-generated EMVs may contribute to the proinflammatory vascular effects of insulin.

Nothing to disclose

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.
The relationship between subjective pain ratings and sympathetic outflow during a cold pressor test
Authors: Chaudhry Laila , Coovadia Yasmine , Chen Celine , Usselman Charlotte
Published: May 2023

Pain is the leading source of morbidity worldwide and a global clinical and societal challenge. Subjective pain scores are commonly employed in pain-centric research to quantify pain despite the presence of established organismic and environmental sources of intra- and interindividual variability in these scores. As a result, methods for the quantification of pain continue to be the focus of ongoing work in the pain field, including the search for objective biomarkers for pain.To that end, muscle sympathetic nerve activity (MSNA) has been proposed as an objective biomarker of pain. Therefore, the purpose of this study was to examine the relationship between changes in MSNA and changes in subjective pain ratings (PR) during an acute pain stimulus (i.e., cold pressor test (CPT)). Additionally, we examined whether MSNA measures were sensitive to within-subject changes in PR across time. We hypothesized that MSNA would be positively correlated with PR, and that both would increase steadily and concomitantly throughout a 6-minute CPT. We obtained integrated MSNA using peroneal microneurography (NeuroAmp, ADI; MSNA quantified as burst incidence (bursts/100hb)) during 10 minutes of supine rest and CPT in 10 young (aged 18-35 y) and healthy individuals (n=5 men). Participants reported PR on a numerical rating scale (0-10) at rest and during the CPT (timepoints: 30-s, 120-s, 240-s, and 330-s). One-way repeated measures ANOVAs assessed whether PR and MSNA changed over the CPT. PR increased over the CPT (p= 0.03) with a mean increase of 1±3 rating scale points from 30-s to 330-s. There was no main effect of time on MSNA (p=0.051), as it increased initially, but then decreased gradually thereafter. A linear regression analysis of the relationship between absolute PR and MSNA demonstrated a moderate positive correlation (r=0.43, p<0.01); however, relative changes in PR and MSNA over time (i.e., timepoints expressed relative to rest) were not correlated (r=-0.19, p=0.25). Meanwhile, acute changes in MSNA and PR from timepoint-to-timepoint (calculated as timepoint - previous timepoint) were strongly correlated (r=0.62, p<0.01). In summary, we observed similar patterns of change in subjective PR scores and MSNA during a CPT. These data suggest that MSNA is a valuable biomarker in the quantification of pain due to its consistency with subjective measures of pain.

This research was funded by the National Science and Engineering Research Council of 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.
Use of the Peregrine Catheter Infusion System to develop novel targeted renal nerve ablation therapies in hypertensive sheep
Authors: de la Cruz-Lynch Arthur , Van Helden Dusty , Dayton Alex , Evans Louise , Ruiz Lauar Mariana , Osborn John
Published: May 2023

Background and Objective: Clinical trials have shown that catheter-based total (efferent and afferent) renal denervation (TRDN) can effectively lower arterial pressure in patients with treatment-resistant hypertension (HTN). The Peregrine catheter infusion system deploys microneedles from the renal artery lumen into the renal artery adventitia to inject the neurolytic agent ethanol. The objective of this study was to utilize this catheter to develop novel renal denervation therapies in a large animal model. In this study our aim was to demonstrate that TRDN lowers arterial pressure in the deoxycorticosterone acetate and high salt diet (DOCA-salt) sheep model of HTN. This will establish the utility of this catheter to deliver the afferent nerve neurolytic agent, capsaicin. Our group has shown that selective afferent renal denervation (ARDN) lowers arterial pressure to the same degree as TRDN in DOCA-salt HTN rats. Our long-term goal is to translate these findings to the DOCA-salt sheep model and eventually humans. Methods: Adult Domestic sheep weighing 60-80 kg were implanted with a subcutaneous silicone pellet containing DOCA (350mg/kg) and given access to salted (0.2% NaCl) drinking water and (180mEq Na+/kg) feed. Stellar radio telemeters were implanted for continuous measurement of MAP. 30 days following induction of DOCA-salt HTN, the Peregrine catheter infusion system was used to perform bilateral TRDN. One month after TRDN, animals were terminated and kidneys were harvested to verify denervation efficacy. Preliminary Results: 30 days following implantation of DOCA, 24hr MAP increased ~25mmHg from a baseline of 99mmHg to 123mmHg (n=2). 30 days following TRDN, 24hr MAP decreased to near control levels of 105mmHg. Conclusion and Future Directions: These preliminary results demonstrate that TRDN with the Peregrine catheter lowers MAP in DOCA-salt HTN sheep. The mechanism underlying this response is still being investigated. Sheep studies are underway to utilize the Peregrine catheter to perform ARDN by injection of capsaicin into the renal artery adventitia. The goal is to conduct a translational study to move catheter-based ARDN into clinical trials. If catheter-based ARDN lowers MAP to the same degree as TRDN, efferent renal nerves could be preserved to maintain MAP and blood volume in response to hemorrhagic or septic shock.

Supported by NHLBI R01 HL116476.

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.
The cellular circadian clock of Phox2b-expressing cells drives daily variation of the hypoxic but not hypercapnic ventilatory response in mice
Authors: Jones Aaron , Marino Gabriella , Spears Allison , Arble Deanna
Published: May 2023

While the suprachiasmatic nucleus (SCN) controls 24-hour rhythms in breathing including minute ventilation (VE), the mechanisms by which the SCN drives these daily changes are not well-understood. Moreover, the extent to which the circadian clock regulates the ventilatory responses to hypercapnia and hypoxia (i.e., the ventilatory chemoreflex), is unknown. We hypothesized that the SCN regulates daily breathing and chemoreflex rhythms by synchronizing the molecular circadian clock within cells. Here, we used whole-body plethysmography to assess ventilatory function in transgenic BMAL1 knockout (KO) mice to determine the role of the cellular clock in regulating daily rhythms in ventilation and chemoreflex. We observed that, unlike their wildtype littermates, BMAL1 KO mice exhibited a blunted daily rhythm in VE and failed to demonstrate daily variation in the hypoxic ventilatory response (HVR). The absence of a functional cellular clock in BMAL1 KO mice also resulted in an overall decrease in the hypercapnic ventilatory response (HCVR) and a sex-specific absence of circadian variation of HCVR. Next, to determine the extent to which these effects on minute ventilation and ventilatory chemoreflex were mediated by the cellular clock within the neural respiratory network, we assessed ventilatory rhythms in BMAL1fl/fl;Phox2bCre/+ mice which lack BMAL1 in all Phox2b-expressing chemoreceptor cells (hereafter called BKOP). BKOP mice lacked daily variation in HVR consistent with our observations in the BMAL1 KO mice. However, unlike BMAL1 KO mice, BKOP continued to exhibit daily variations in VE and HCVR similar to wildtype littermates. These data indicate that the cellular clock contributes to the daily rhythms in VE, HVR, and HCVR. Moreover, the cellular clock of Phox2b-expressing cells specifically is necessary for day-night variations in HVR. Taken together, these data suggest that the SCN regulates daily ventilatory rhythms in part through the synchronization of cellular clocks within respiratory cell populations. These findings open the possibility that circadian disruption or circadian misalignment may affect respiratory homeostasis which, in turn, may have clinical implications for shift workers and respiratory diseases with time-of-day-specific symptomology.

American Heart Association (Arble, 20IPA35320195)

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.
Pannexin 1 channels control CaV1.2 channel activity and vascular reactivity in diabetic hyperglycemia
Authors: Hong Junyoung , Hong Junyoung , Baudel Miguel Martin-Aragon , Reddy Gopyreddy R , Moustafa Yumna A , Ayende Jose W , Mohabbat Yaseen , Isakson Brant E , Nieves-Cintron Madeline , Navedo Manuel F
Published: May 2023

Vascular complications are major causes of morbidity and mortality in diabetic patients. Recently, our group identified a novel purinergic signaling pathway involving the AKAP5/P2Y11/AC5/PKA/CaV1.2 protein network in arterial myocytes that is activated upon diabetic hyperglycemia leading to vasoconstriction. This purinergic complex is activated by the release of nucleotides (e.g. ATP) to the extracellular space in response to hyperglycemic conditions. Pannexin 1 (Panx1) is a channel that mediates ATP efflux, thus inducing purinergic signaling activation. In this study, we hypothesize that Panx1 is a critical regulator mediating ATP release and increasing cAMP synthesis, CaV1.2 potentiation, and vasoconstriction upon diabetic hyperglycemia. Consistent with this hypothesis, we found that elevated glucose (HG) induced an elevation in extracellular ATP concentrations, and this was reduced in the presence of the Panx1 inhibitor spironolactone (spiro). Panx1 was found in complex with AKAP5, P2Y11, AC5, PKA, and CaV1.2 in arterial myocytes. This protein complex was strengthened upon HG treatment, and spiro prevented this effect. Spiro and genetic ablation of smooth muscle Panx1 (iPanx1sm -/-) blocked cAMP production, CaV1.2 potentiation, sustained vasoconstriction, and in vivo elevations in cerebral artery myogenic tone and reduced blood flow in response to HG. In a mouse model of type 1 diabetes (e.g. STZ), the increased CaV1.2 potentiation and enhanced myogenic tone were prevented in arterial myocytes and arteries from iPanx1sm -/- mice. Altogether, these data suggest that Panx1 is part of the AKAP5/P2Y11/AC5/PKA/CaV1.2 signaling module in arterial myocytes. This Panx1-led complex modulates CaV1.2 activity and vascular reactivity in response to diabetic hyperglycemia. Thus, Panx1 could be a new therapeutic target to treat vascular complications during diabetes.

This work was supported by NIH grants R01HL149127, R01HL121059, and R01HL161872 (to MFN).

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.
Exercise Hyperemia Following Ischemia-Reperfusion
Authors: Hanson Brady , Miller Kayla , Casey Darren , Hanada Satoshi , Feider Andrew , Aldrich Aric
Published: May 2023

Prolonged ischemia can result in cellular dysfunction. Paradoxically, the reintroduction of blood flow (reperfusion) can lead to further damage, described as ischemia-reperfusion injury (I/R). It has been reported that I/R attenuates endothelial function at the micro- and macrovascular level. An attenuation in microvascular endothelial function from I/R may therefore disrupt the ability to modulate exercise hyperemia in contracting skeletal muscle. We tested the hypothesis that I/R blunts the rapid and steady-state hyperemic and vasodilatory response to rhythmic handgrip exercise. Ten young healthy subjects (8M/2F; 24 ± 1 yr) completed two separate study visits (I/R and time control trials) in a random order. On each study visit rapid-onset hyperemia and vasodilation following a single forearm contraction was assessed in duplicate. Rhythmic handgrip exercise (20 contractions per min) was then completed for 3 min. All handgrip exercises were performed at 20% maximum voluntary contraction. Following exercise, either I/R was induced (pneumatic cuff placed around the upper left arm at 240 mmHg for 20 min followed by 20 min of reperfusion) or a time control trial (supine rest for 40 min) was completed. Handgrip exercises were then repeated immediately after each respective intervention. Brachial artery blood velocity and diameter were measured with Doppler ultrasound and forearm vascular conductance (FVC) was calculated as forearm blood flow (FBF; ml·min-1) over blood pressure (mmHg). In response to a single muscle contraction, peak hyperemia (ΔFBF from baseline) and vasodilation (ΔFVC) were attenuated following I/R (134 ± 15 vs. 103 ± 13 ml·min-1; 160 ± 17 vs. 118 ± 15 ml·min-1·100 mmHg-1, P<0.05 for both) but not following time control (115 ± 20 vs. 124 ± 18 ml·min-1; 150 ± 25 vs. 148 ± 20 ml·min-1·100 mmHg-1, P=0.16 and P=0.95, respectively). Total FBF and FVC following a single muscle contraction was not different between I/R (25 ± 4 vs. 20 ± 3 ml; 33 ± 5 vs. 24 ± 4 ml·100 mmHg-1) and time control (21 ± 4 vs. 23 ± 4 ml; 28 ± 5 vs. 28 ±4 ml·100 mmHg-1) trials (group x time interactions P=0.05 and P=0.08, respectively). Steady-state ΔFBF and ΔFVC during rhythmic exercise was unchanged in both I/R (192 ± 16 vs. 190 ± 17 ml·min-1; 208 ± 18 vs. 193 ± 19 ml·min-1·100 mmHg-1) and time control (188 ± 17 vs. 196 ± 15 ml·min-1; 206 ± 19 vs. 207 ± 15 ml·min-1·100 mmHg-1) trials (group x time interactions P=0.34 and 0.21, respectively). Our data indicate that 40 min of I/R blunts hyperemia and vasodilation at the onset of skeletal muscle contraction, but does not appear to attenuate these responses during steady-state rhythmic exercise.

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.
Muted molecular responses to thermal stress in the Antarctic emerald rockcod (Trematomus bernacchii): Proteomic evidence and possible evolutionary mechanisms
Authors: Dowd Wes , Kueltz Dietmar
Published: May 2023

The Antarctic emerald rockcod ( Trematomus bernacchii) and its relatives have long been of interest to comparative physiologists, given their history of isolation in extremely cold waters and indications that they lack some or all of the classic “heat shock response.” In this study we complemented previous transcript-level analyses with a comprehensive assessment of the proteomic response to elevated temperatures across three tissues (gill, head kidney, and brain). Animals were exposed to constant conditions at one of three acclimation temperatures, after which a subset were sampled and the remaining individuals experienced an acute heat stress with or without recovery (n=5-6 per treatment). Protein expression patterns were analyzed using tissue-specific, data-independent acquisition (DIA) assay libraries on a UPLC-MS system. The expression patterns reveal a muted response to these different thermal challenges, with only a small number of proteins (out of 2721-3421 per tissue) differentially expressed for any given tissue-treatment combination. The magnitudes of expression changes were also small. These results appear to contradict relatively robust induction patterns for a subset of cellular stress response genes obtained from previous transcriptomic analyses. Furthermore, the differentially expressed proteins were not functionally enriched for any Gene Ontology or KEGG database entries, nor were the promoter regions of the corresponding genetic loci consistently enriched for any transcription factor binding motifs, including those that might be predicted to play a role in the response to hyperthermal stress (such as Hsf1 or HIF). Subsequent bioinformatics approaches within the Perciformes clade suggest modifications to the suite of proteins that regulate translation as a plausible explanation for the lack of a coordinated response to thermal stress in the Antarctic lineage. Overall, our proteomics data offer further detail into the physiological underpinnings of stenothermy in Antarctic fishes.

NSF IOS-165582 to WD and an APS John F. Perkins, Jr., Research Career Enhancement Award

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.
The effect of sex on sympathetic transduction during an acute pain stimulus
Authors: Coovadia Yasmine , Chaudhry Laila , Fraser Graham , Usselman Charlotte
Published: May 2023

Previously, we have noted sex differences in beat-by-beat fluctuations in blood pressure following sequences of muscle sympathetic nerve activity (MSNA; i.e. sympathetic transduction) during acute sympatho-inhibition but not at rest. However, sex differences in sympathetic transduction during sympatho-excitation have yet to be examined. Therefore, we tested the hypothesis that sympathetic transduction during acute sympatho-excitation and recovery from sympatho-excitation are greater in men than women. We tested healthy women (n=5) and men (n=5) of similar age (26±4 vs 23±2y, respectively; P=0.3). Integrated MSNA (microneurography; NeuroAmp, ADInstruments) and beat-by-beat blood pressure (finger photoplethysmography; Finometer MIDI) were assessed during 10 minutes of supine rest, an acute pain stimulus (6-minute cold pressor test; CPT), and recovery (6-minutes). Custom software was used to calculate sympathetic transduction: beat-by-beat changes in mean arterial pressure (MAP) were tracked for 15 subsequent cardiac cycles (expressed relative to the cardiac cycle associated with the final burst or non-burst in the sequence). Like sequences were overlaid and averaged. Prevailing MSNA and MAP were not different between women and men at rest (9±8 vs 8±5 bursts/min, P=0.8; 88±6 vs 97±6 mmHg, P=0.06) or during the CPT (21±10 vs 16±6 bursts/min, P=0.3; 98±5 vs 104±6 mmHg, P=0.1). However, during recovery, MSNA was greater in women than men (21±8 vs 10±4 bursts/min, P=0.03), despite similar MAP (95±5 vs 99±6 mmHg, P=0.3). Peak and nadir changes in sympathetic transduction following burst and non-burst sequences were not different between women and men at rest (burst: 1.55±0.46 vs 1.67±0.27mmHg, P=0.6; non-burst: -2.21±1.88 vs -2.26±0.36mmHg, P=0.9) or during the CPT (burst: 1.06±0.32 vs 0.45±0.47mmHg, P=0.08; non-burst: -3.24±1.44 vs 1.90±0.46mmHg, P=0.1). Sympathetic transduction following a burst of MSNA did not differ between the sexes during recovery (1.73±0.34 vs 1.65±0.23mmHg, P=0.7), while nadir decreases in MAP following non-burst sequences during recovery were more pronounced in women than men (-2.81±0.75 vs -1.56±0.91mmHg, P=0.04). Taken together, although sympathetic transduction did not differ between the sexes during the CPT, these data suggest that women may require heightened MSNA during recovery from acute stress to maintain equivalent blood pressure.

This project was funded by McGill University, FRQS, and NSERC.

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.
Cldn2-Trpv6 double knockout mice have hypocalcaemia and reduced bone mineral density
Authors: Bhullar Harneet , Alexander Todd
Published: May 2023

Central to calcium homeostasis and bone health is adequate intestinal calcium absorption. This occurs via either a transcellular or a paracellular pathway. In cases of low dietary calcium availability, a transcellular pathway that relies at least in part on the apical calcium channel Trpv6 predominates. However, when calcium is abundant in the diet, paracellular calcium absorption occurs through tight junction proteins, including claudin-2 (Cldn2). This tight junction protein also plays a role in calcium reabsorption from the renal proximal tubule. Despite this, Cldn2 knockout mice only display mildly altered calcium homeostasis characterized by hypercalciuria without evidence of altered bone mineral density (BMD), plasma calcium or calciotropic hormone levels. Similarly, mice expressing non-functional mutant Trpv6 (TRPV6D541A/D541A) channels have reduced intestinal calcium absorption without altered urine or plasma calcium levels, and only when fed a low calcium diet. We hypothesized that the lack of a more pronounced phenotype in these single functional knockout mice was due to the compensation of one pathway in the absence of the other. To test this hypothesis, we crossed claudin-2 knockout mice with mice expressing the mutant Trpv6, generating a functional double knockout (dko, Trpv6mut/mut/Cldn2-/- mice). Metabolic cage studies revealed hypocalcaemia (Male WT: 1.07 ± 0.02 mM, Male DKO: 0.97 ± 0.02 mM, p-value=0.0065, Female WT: 1.07 ± 0.02 mM, Female dko: 0.96 ± 0.03 mM , p-value= 0.0050) and hypercalciuria (Male WT Ca2+/Creatinine: 0.51 ± 0.05, Male dko:1.93 ± 0.18, p-value < 0.0001 Female WT Ca2+/Creatinine: 1.10 ± 0.13, Female dko: 3.56 ± 0.28 , p-value < 0.0001) in the dko mice. In contrast to the single claudin-2 KO mice and Trpv6 mutant animals the dko mice had elevated plasma calcitriol and parathyroid hormone. Moreover, BMD was reduced in the dko animals (Cortical BMD WT: 1.36 ± 0.02 g/cm3 vs dko: 1.29 ± 0.02g/cm3 ). RT-qPCR of intestinal segments and whole kidney revealed increased calbindin-D9k expression in the proximal colon (but not duodenum) and calbindin-D28k in the kidneys, consistent with compensatory increased transcellular absorption from these intestinal segments. Together these results are consistent with the paracellular pathway compensating for the loss of transcellular calcium absorption from the intestine.

Funding: Alberta Innovates Graduate Studentship, NSERC, CIHR

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.
Effects of acute intermittent hypoxia on P0.1 in humans with spinal cord injury
Authors: Mir Michela , Vose Alicia , Brunetti Gina , Cavka Kathryn , DeMark Louis , Snyder Hannah , Wauneka Clayton , Welch Joseph , Nair Jayakrishnan , Mitchell Gordon , Fox Emily
Published: May 2023

Spinal cord injury (SCI) disrupts neural pathways to respiratory motor neurons, diminishing respiratory drive and breathing function. Breathing impairment remains a leading cause of illness and death after SCI. Therapeutic acute intermittent hypoxia (tAIH; 15, 1-minute episodes of 9% inspired O2, 1.5 min normoxic intervals), a promising strategy to restore breathing function after SCI, promotes spinal neuroplasticity in rodent models. However, in humans, respiratory drive is unchanged after a single tAIH session; thus we tested the hypothesis that tAIH on five successive days increases neuromechanical respiratory drive in humans living with chronic SCI. Sixteen participants with chronic SCI (1 female; 33.5 + 15.3 years of age; 1-28 years post-SCI; 11 cervical, 5 thoracic; C1-T6 AIS A-D) were studied; their respiratory function was 52% ± 19% of age predicted expiratory pressure generation, and 123% ± 32% for inspiratory pressure. Five days of tAIH or Sham treatment were delivered in a randomized, cross-over design (minimum 3 weeks apart). Airway occlusion pressure in the first 0.1s of inspiration (P0.1), a surrogate for neuromechanical respiratory drive prior to within-breath sensory feedback, was collected at baseline and at 1, 3, and 7-days post-treatment. Repeated measures ANOVA revealed no significant effect of time (F(3)=.49; P=.69) or group (F(3)=1.33; P=.27). Despite the lack of significant differences in this preliminary analysis, P0.1 increased by 25% on day 3 versus 0.8% after Sham. Because of this change, pairwise comparisons were conducted, revealing a near significant increase in P0.1, 3 days post tAIH (MD=-.17; P =.06). Thus, although P0.1, a surrogate for neuromechanical respiratory drive, did not significantly increase after 5 days of AIH in people with SCI, the marginally significant change at day 3 suggests further investigation is warranted. Next steps include assessment in a larger sample, investigation of biomarkers for treatment effects, and examination of combinatorial interventions including task specific training and/or mild CO2 elevations to enhance AIH-induced neuroplasticity in the drive to breathe.

DoD SCIRP Clinical Trial Award W81XWH-17-SCIRP-CTA; The Craig H. Neilsen Foundation (JW); NIH T32 HL134621 (MM); and the UF McKnight Brain Institute.

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.
The role of short-chain fatty acid receptors in the hypothalamic paraventricular nucleus of adult offspring in hypertension programmed by maternal high fructose diet exposure
Authors: Chan Julie Y.H.
Published: May 2023

The role of short-chain fatty acids (SCFAs) in the brain on developmental programming of hypertension is poorly understood. The present study aimed to explore the dysregulated tissue levels of SCFAs and expression of SCFA-sensing receptors in the hypothalamic paraventricular nucleus (PVN), a key forebrain region engaged in neural regulation of blood pressure, of offspring to maternal high fructose diet (HFD) exposure. We further investigated the engagement of SCFA-sensing receptors in the PVN on the beneficial effects of -biotics (prebiotic, probiotic, synbiotic and postbiotic) on programmed hypertension primed by maternal HFD. Maternal HFD during gestation and lactation significantly reduced circulating butyrate, along with decreased tissue level of butyrate and increased expression of the SCFA-sensing receptors, GPR41 and olfr78, in the PVN of HFD offspring that could be rectified by oral supplement with prebiotic, probiotic, or synbiotic. The maternal HFD-primed programmed hypertension in adult offspring was accompanied by tissue oxidative stress and neuroinflammation in the PVN, both of which were reversed by oral supplement with the -biotics. Gene silencing of GPR41 or olfr78 mRNA in the PVN also protected adult HFD offspring from programmed hypertension and alleviated the induced oxidative stress and inflammation in the PVN. In addition, oral supplement with the postbiotic butyrate notably restored tissue butyrate level and rectified the expressions of GPR41 and olfr78 in the PVN of HFD offspring. The same treatment also protected against programmed hypertension in adult HFD offspring. These data together suggest that alterations in tissue butyrate level and expression of the SCFA-sensing receptors of GPR41 and olfr78, as well as activation of the SCFA-sensing receptor-dependent tissue oxidative stress and neuroinflammation in the PVN could be novel mechanisms that underlie hypertension programmed by maternal HFD exposure in adult offspring. Furthermore, oral -biotics supplementation may exert beneficial effects on hypertension of developmental origin by targeting the dysfunctional SCFA-sensing receptors in the PVN to exert antioxidant and anti-inflammatory actions in the brain.

This research was funded in part by the Chang Gung Medical Foundation, Taiwan (grant number: OMRPG8G0011), and the Ministry of Science and Technology, Taiwan (grant number: MOST111-2320-B-182A-015).

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.
Mitochondrial integrity distinguishes exercise-induced vs. pathogenic cardiac hypertrophy
Authors: Yang Nicole Lee , Strumwasser Alexander , Zhou Zhenqi , Moore Timothy , Hevener Andrea
Published: May 2023

Cardiac hypertrophy is a consequence of exercise training, as well as certain cardiomyopathies. However, the key genetic drivers of cardiac remodeling in a healthy vs diseased heart are inadequately understood. Our purpose was to determine the genetic architecture of exercise training-induced cardiac remodeling and hypertrophy. The exercise hybrid mouse diversity panel (ExcHMDP) is comprised of 100 strains of Exc trained (TRN) and sedentary (SED) animals (n=4-8/strain/group). After 30d of exercise, running wheels were locked, and 24-hours post-exercise, 6h-fasted animals were euthanized and tissues harvested (~20 tissues/mouse). To interrogate pathogenic cardiac hypertrophy, isoproterenol (ISO, 30mg/kg/day for 21d) was administered to a second HMDP cohort, and similar to TRN hearts, RNAseq analyses were performed. TRN increased heart weight in 85 of 100 mouse strains vs. SED. Of the nearly thirty heart phenotypes assessed, none correlated significantly with running distance. Enrichment analysis of differential gene expression revealed mitochondrial function, inflammatory and immune processes, calcium signaling, muscle growth and development, and angiogenesis (FDR<0.05) as top pathways impacted by TRN. Biological processes divergent between the Exc and ISO-HMDPs included oxidative phosphorylation, electron transport chain, and mitochondrial respiratory chain complex assembly (FDR<1.1E-07). Electron microscopy analyses of TRN vs. ISO hearts showed differential remodeling of mitochondrial cristae involved in oxidative metabolism. Preliminary sex differences studies using an acute cardiac Esr1 (estrogen receptor alpha, ERa) knockout revealed reduced cardiac function, impaired mitochondrial metabolism, and extensive myocardial fibrosis in adult female C56Bl6/J mice (n=6); molecular assays revealed increased expression of alpha smooth muscle actin (P=0.002) and collagens Col1a2 and Col6a3 (P=0.02; P=0.006), suggesting that ERa may be a mediator of sex-specific cardiac remodeling. Our studies provide important insight into the genetic architecture of cardiac remodeling associated with healthy vs. pathogenic hypertrophy. A primary goal is that our transcriptomics analyses are leveraged to advance therapeutics to combat hypertrophic cardiac myopathy-associated heart failure.

Funding: NIH U01 AG070959; U54DK120342

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.
FOXO1 cooperates with C/EBPΔ and ATF4 to regulate skeletal muscle atrophy transcriptional program during fasting
Authors: Kamei Yasutomi , Oyabu Mamoru
Published: May 2023

Catabolic conditions, such as starvation, inactivity, and cancer cachexia, induce Forkhead box O (FOXO) transcription factor(s) expression and severe muscle atrophy via the induction of ubiquitin–proteasome system-mediated muscle proteolysis, resulting in frailty and poor quality of life. Although FOXOs are clearly essential for the induction of muscle atrophy, it is unclear whether there are other factors involved in the FOXO-mediated transcriptional regulation. As such, we identified FOXO–CCAAT/enhancer binding protein Δ (C/EBPΔ) signaling pathway as a novel proteolytic pathway. By comparing the gene expression profiles of FOXO1-transgenic (gain-of-function model) and FOXO1,3a,4–/–(loss-of-function model) mice, we identified several novel FOXO1-target genes in skeletal muscle including Redd1, Sestrin1, Castor2, Chac1, Depp1, Lat3, as well as C/EBPΔ. During starvation, C/EBPΔ abundance was increased in a FOXOs-dependent manner. Notably, knockdown of C/EBPΔ prevented the induction of the ubiquitin–proteasome system and decrease of myofibers in FOXO1-activated myotubes. Conversely, C/EBPΔ overexpression in primary myotubes induced myotube atrophy. Furthermore, we demonstrated that FOXO1 enhances the promoter activity of target genes in cooperation with C/EBPΔ and ATF4. This research comprehensively identifies novel FOXO1 target genes in skeletal muscle and clarifies the pathophysiological role of FOXO1, a master regulator of skeletal muscle atrophy.

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.
Identification of protein machineries involved in aquaporin-2 gene transcription with genomic locus proteomics (GLoPro)
Authors: Lin Yi-Jiun , Yeh Ai-Hsin , Chen Hsuan-I , Liu Chin-Fu , Yu Ming-Jiun
Published: May 2023

Vasopressin-induced water reabsorption by the renal collecting ducts is in part regulated by aquaporin-2 gene expression; however, the mechanism is not completely understood. We used genomic locus proteomics (GLoPro) to identify transcription protein network in the aquaporin-2 5’-flanking region in the mpkCCD cell model. Our strategy relied on a recombinant protein consisted of catalytic dead Cas9 and biotin ligase TurboID (dCas9-TurboID). When guided to -95 bp upstream to the aquaporin-2 transcription start site, it did not affect vasopressin analog dDAVP-induced aquaporin-2 gene transcription. The dCas9-TurboID was used to biotinylate proteins recruited by dDAVP to the above genomic region before affinity-purification followed by label-free identification and quantification with LC-MS/MS. In three independent experiments, 1,647 and 1,676 proteins were identified respectively in the vehicle and dDAVP-treated cells. Bioinformatics analysis showed enriched cellular component GO terms: nucleus, nucleoplasm, and nucleolus, consistent with functions of these identified proteins in gene regulation. Statistical analysis showed up-regulation of 310 proteins and down-regulation of 254 proteins in response to dDAVP. Biological process GO term analysis showed enrichment for mRNA processing, RNA splicing, and chromatin organization. In the dDAVP-upregulated group, we identified proteins involved in chromatin remodeling and histone modification that adjust chromatin accessibility: Baz2b, Npm3, Cbx8, Cdk17, Meaf6, and Prmt1. We also identified transcription factors and cofactors: Paxbp1, Rbm14, Cxxc1, Rbpms, Ccar1, Chch2, and JunB. Of these, JunB has been implicated in aquaporin-2 gene transcription. Based on prior omics data, we used Bayes’ theorem to rank these proteins for possibility in aquaporin-2 gene transcription. The top candidate was Ccar1, which is known to complex with β-catenin and T cell factor to enhance target gene transcription. Vasopressin induces β-catenin nuclear translocation; T cell factor has a conserved binding element in the aquaporin-2 5’-flanking region. Further study is needed to examine the possibility that Ccar1 involves β-catenin and T cell factor to regulate aquaporin-2 transcription.

This research was supported by National Science and Technology Concil, R.O.C. (MOST111-2320-B-002-084-MY3).

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.
Cardiovascular and respiratory profile of adenosine A 2A receptors knockout mice submitted to sustained hypoxia
Authors: Reis Souza Juliana , Honório Machado Benedito
Published: May 2023

Taking into consideration the relevance of adenosine as a signaling molecule during hypoxic challenges and the evidence of an important role of A2A receptors regions of the brainstem involved with the generation and modulation of the autonomic and respiratory functions, in the present study we evaluated the cardiovascular and respiratory changes in response to sustained hypoxia (SH) in knockout mice for A2A receptors (A2AKO). For this, we used male A2AKO and male Balb/c wild-type (WT) mice as a control. Under anesthesia, catheters were inserted into the femoral artery, for recording arterial pressure (MAP), and jugular vein for drug injection. Four days after, mice were submitted to normoxia or SH (24h, FiO2 0.1). At the end of these protocols, cardiovascular and respiratory parameters were recorded. Peripheral chemoreflex was activated by potassium cyanide (KCN, 0,16 mg/Kg, iv). In distinct groups of A2AKO and WT mice, with the femoral artery previously catheterized, respiratory parameters were evaluated before and after SH using whole-body plethysmography. At the end of respiratory recordings, a sample of arterial blood was collected for arterial blood gases analysis. Similar and ignificant reductions in MAP and heart rate (HR) were observed in WT [(102±5 vs 109±6 mmHg, P=0.0172), (329±31 vs 441±55 bpm, P<0.0001)] and A2AKO [(104±5 vs 111±7 mmHg; P=0.0141), (318±59 vs 408±64 bpm, P=0.0006)] mice submitted to SH when compared to their respective normoxia controls (WT: n=11; A2AKO: n=13). Mice from control and A2AKO groups presented similar cardiovascular responses to KCN in normoxic as well as in hypoxic conditions. Under normoxic conditions, A2AKO mice (n=11) presented significant increase in respiratory frequency (241 ± 25 vs 184 ± 11 breaths.min-1, P < 0.0001) when compared to respective genetic controls (n=8). Both A2AKO [(24±3 vs 32± 2 mmHg; P<0.0001), (105±6 vs 90±4 mmHg, P = 0.0002)] and WT [(23±4 vs 32±3 mmHg, P<0.0001), (100±9 vs 88±10 mmHg, P=0.0034)] mice presented a significant reduction in pCO2 and an increase in pO2 after SH. The data are showing that the lack of adenosine A2A receptors does not affect the autonomic responses to chemoreflex activation, but are relevant in modulating respiratory activity in normal mice since in the A2AKO mice it was observed a significant increase in respiratory frequency. The data also suggest that A2A receptors play no major role in autonomic and respiratory changes in mice submitted to SH.

FAPESP (2018/15957-2 and 2021/01767-0) and CNPQ (309338/2020-4).

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.
TUDCA treatment restores early protein-restriction-induced aortic hipocontractility and endothelial dysfunction
Authors: Freitas Israelle , Junior Joel , Victorio Jamaira , Carneiro Everardo , Davel Ana Paula
Published: May 2023

TUDCA treatment restores early protein-restriction-induced aortic hipocontractility and endothelial dysfunction Israelle Netto Freitas1,2; Joel da Silva Junior2; Jamaira Aparecida Victorio1,2; Everardo Magalhães Carneiro1,2; Ana Paula Davel1 1Laboratory of Vascular Biology and 2Obesity and Comorbidities Center-OCRC, Institute of Biology, University of Campinas-UNICAMP, Campinas, SP, Brazil. Malnutrition is a risk factor for the development of vascular dysfunction. Tauroursodeoxycholic acid (TUDCA) inhibits endoplasmic reticulum stress (ERS) and has been demonstrated to be protective against vascular dysfunction in cardiometabolic diseases. Therefore, we hypothesized that TUDCA could be beneficial for endothelial function and vascular contractility during malnutrition. For this, post-weaning male and female mice fed a normoprotein (NP, 14% protein) or low protein (LP, 6% protein, isocaloric) diet for 15 weeks. In the last 2 weeks, some NP and LP animals received TUDCA (300 mg/kg/day, ip) or vehicle. At the end of the treatment, body weight (BW) and systolic blood pressure (SBP) were evaluated and thoracic aortic isolated to assess vascular responses, fluorescence to dihydroethidium (DHE) and gene expression. As results, BW was reduced and SBP was elevated in male and female protein-restricted groups; TUDCA treatment restored SBP but not BW. Only in males, protein restriction impaired endothelium-dependent relaxation to acetylcholine and reduced contractility (phenylephrine, serotonin, and TXA2 analogue U46619) in aorta. TUDCA reversed both endothelial dysfunction and hypocontractility in LP male group. We found reduced expression of smooth muscle 22α (SM22α), α-actin and L-type calcium channel (Cav1.2) in aorta from male LP compared to NP, which was restored by TUDCA treatment. In addition, aorta from male LP group showed increased DHE fluorescence and gene expression of the ERS markers GRP78, ATF6, PERK, IRE1α and CHOP. These alterations were reversed by TUDCA. In conclusion, our data suggest that post-weaning protein restriction results in endothelial dysfunction and aortic hypocontractility in males only. These alterations were associated with oxidative stress and reduced expression of vascular contractile and ERS markers. TUDCA treatment rescued vascular contraction and endothelial function in early protein-restricted mice evidencing the therapeutic potential of this bile acid for the vascular complications associated with undernutrition.

The authors have nothing to disclose. Funding source: São Paulo Research Foundation (FAPESP).

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.