HTML Format Research Articles

An Innovative Apical-4-Chamber-long-axis Imaging Approach Precisely Tracks Progressive Deterioration of Mouse Ventricles, Atria, and Atrioventricular Valves

Transthoracic echocardiography stands as the primary non-invasive method for cardiac function evaluation in clinical settings. It’s widely used to assess chamber sizes, heart wall integrity, as well as the hemodynamic performance of the left ventricle (LV), right ventricle (RV), atria, and valves. However, applying comprehensive echocardiography assessments to mice has been challenging, primarily due to diffculties in obtaining on-axis apical 4-chamber views; most mouse studies have thus been restricted to evaluating the LV using short-axis M-mode assays. To overcome this limitation, we’ve recently developed a novel methodology to consistently obtain high-quality apical 4-chamber-long-axis (A4CLAX) view images of mouse heart. This advancement permits a range of clinically relevant functional assessments, including Long Axis Biplane (LAX-BP) assay, which has been shown to surpasses M-mode in estimating LV function. In this study, we delve into the breadth of comprehensive echocardiography assessments made possible for mice through the A4CLAX imaging approach. We performed a severe transverse aortic constriction (TAC) surgery on mice and monitored the pathological development in their hearts post-surgery. Alongside the expected hypertrophy, the LAX-BP assay revealed LV volumetric dilation as early as 1-week after TAC surgery Indicative of decompensated heart failure; however, m-mode was not able to pick up on this early dilation due to its lack of longitudinal assessment and inconsistent measurements of EDV and ESV. The apical 4 chamber view significantly improved the consistency and reliability of trans mitral and pulmonary vein doppler recordings and enabled us to reliably estimate the pressure relationships of the LV and Left Atrial (LA) pressure, which is consistent with the LAX-BP detected LV decompensation. Additionally, the high clarity of the 4 chamber images achieved improved visualization of both mitral and tricuspid valve leaflets. This allowed more precise characterization of valvular issues, including mitral regurgitation caused by LV dilatation, as well as identification of less common pathological occurrences like desynchronization of the tricuspid and mitral valve opening. A speckle tracking assay of the Right ventricle (RV) showed that a delayed contraction of the lateral apical RV wall caused the tricuspid valve to not open during the early filling phase, but rather at the atrial kick, leading to this valve desynchrony. This delayed contraction indicates a possible elevation of pulmonary pressure, as evidenced by pulmonary vein doppler indices and the analysis of pulmonary congestion. In conclusion, the innovative A4CLAX imaging technique revolutionizes murine heart analysis, bringing it in line with clinical echocardiography standards allowing for a whole heart analysis approach. It sensitively identifies subtle anomalies, providing a precise characterization of cardiac remodeling and disease progression in mouse hearts. This study has been supported by grants NIH-R01HL139006 and R21OD031965. This is the full abstract presented at the American Physiology Summit 2024 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 effects of short-term environment-induced heat stress on the myocardium in male mice

Environment-induced heat stress (EIHS) is caused by a sustained elevation in body temperature due to prolonged exposure to excess heat and humidity. Given the current and increasing frequency of EIHS and the large gap in knowledge regarding EIHS-mediated impacts to the myocardium, the purpose of this investigation was to determine the extent EIHS alters cardiac health. We hypothesized EIHS would cause myocardial injury and cellular dysfunction. To test this hypothesis, 13 w old male C57 mice were exposed to EIHS for 4 h or 8 h (n=7; 37.7 ± 0.10 °C, 38.3 ± 0.5 % humidity) or thermoneutral (TN) (n=7, 27.7 ± 0.15 °C, 42.5 ± 0.43% humidity) conditions, hearts were removed, weighed, and portions of the heart were fixed for histology or frozen for biochemical measures. Environment-induced HS increased subcutaneous temperature following 4 and 8 h by 0.9 °C (p< 0.01) compared to respective TN temperatures and decreased body weight by 6.8% (p=0.01) at 4 h and 13% (p<0.01) at 8 h compared to TN. Histological inspection revealed that EIHS increased anisokaryosis by 1.4-fold following 4 h (p<0.01) and by 75% following 8 h (p=0.01) compared to TN animals. Environment-induced HS increased relative abundance of malondialdehyde-modified proteins (35%, p=0.02), a marker of oxidative stress, and increased relative protein abundance of SOD2 (72%, p=0.05) following 4 h compared to TN. Eight hours of EIHS increased relative protein abundance of SOD1 (69%, p=0.01) compared to TN and increased catalase and GPX1 compared to TN (91%, p<0.01; 2-fold, p<0.01) and compared to 4 h of EIHS (72%, p=0.03; 34%, p=0.01), though MDA-modified proteins were similar to TN. Following 4 h of EIHS, markers of mitochondrial abundance, including electron transport chain complexes II (69%, p=0.02) and V (36%, p=0.03), VDAC2 (79%, p=0.01) and PHB1 (65%, p=0.03) were increased compared to TN, and PDH (35%, p=0.04) and Cox IV (55%, p=0.01) were increased compared to 8 hrs of EIHS. Environment-induced HS appeared to alter mitochondrial fusion and fission as DRP1 (51%, p=0.05), phosphorylated (p)-DRP1 (47%, p=0.04), FIS1 (57%, p=0.01) and Mfn1 (26%, p=0.03) were increased compared to TN following 4 h and Mfn2 was increased (37%, p=0.02) following 8 h of EIHS compared to TN. Mitophagy markers BNIP3L/NIX and Pink1 were increased by EIHS following 4 h compared to TN (78%, p=0.02; 41%, p=0.03). Environment-induced HS increased autophagy markers ULK1 (36%, p<0.01), PI3K (80%, p<0.01), and ATG7 (49%, p<0.01) following 4 h compared to TN and ULK1 (33%, p<0.01) and ATG7 (41%, p=0.02) following 4 h compared to 8 h of EIHS. However, p-ULK1, Beclin, p-Beclin, ATG12, and ATG16 were similar between groups. Relative protein abundance of p62 and LC3 II were similar between groups; however, using a colchicine-inhibition approach, 8 h of EIHS caused a significant accumulation of p62, suggestive of increased flux. These data suggest that in a murine model of EIHS, 4 h was suffcient to cause cellular injury and dysfunction in the myocardium; however, as EIHS continued, it was associated with changes reflective of a cellular stress response that may contribute to acclimation. This work was supported by USDA 2020-02716. This is the full abstract presented at the American Physiology Summit 2024 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 GLP-1 Affects Fate of Astrocytes Following Ischemic Stroke in Mice

Stroke is one of the leading causes of physical and intellectual disability and mortality in adults of most of the developed countries. The most of studies in ischemic stroke focus on neuronal pathological mechanisms and neuronal death trying to rescue neurons via stopping the detrimental pathological processes or enhancing neuronal viability. Although astrocytes are the most abundant cells and in mammal central nervous system (CNS), they were ignored for period of time but are return to spotlight in recent years. Astrocyte play multiple roles in maintain normal function of central nervous system and respond to all kinds of CNS injury and disease. Responses of astrocytes to CNS damage lead to both detrimental and beneficial consequences. Previous studies have reported that astrocyte inflammatory responses to ischemic stroke may devastate the ischemic lesion, but other astrocytic responses lead to anti-excitotoxicity, secreting neurotrophic factors, neurogenesis, synaptogenesis, axonal remodeling, and angiogenesis promoting neuroprotective effects. Glucagon-like peptide-1 (GLP-1) have been intensively studied on beneficial effects of neurons, but not many studies work on effects of GLP-1 to astrocytes. Our early study has found that activation of GLP-1 receptors prevented ischemia-induced brain cell death and observed newly neuronal progenitor cells were from astrocyte-lineage in penumbral area of 3-day post-ischemic mouse brains by immunohistochemistry (IHC). RT-PCR, immunoblotting, and immunofluorescent results also clearly indicated the astrocytes expressed GLP-1 receptors. Furthermore, Western blotting results showed administration of GLP-1 analogue, exendin-4 (EX-4), suppressed expression of pro-inflammatory proteins in oxygen-glucose deprivation (OGD) treated mouse primary astrocytes. Interestingly, we also observed that infarct site obtained sever DNA damage and less GFAP positive astrocytes, on contrary, the penumbral area had more GFAP positive astrocytes and less DNA damage in IHC images of stroke brains. The results implied that A1 and A2 astrocytes play certain roles in ischemic brains and recovery. The goal of this study is to determine the mechanisms of GLP-1 involving in astrocytic fate decision and approach on minimizing detrimental effects and amplifying the beneficial effects, which possibly develop innovative therapies for ischemic stroke. The study is supported by 108-2320-B-182A-005-MY3 and 112-2320-B-182A-003- from Taiwan National Science and Technology Council. This is the full abstract presented at the American Physiology Summit 2024 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.

In Vivo Silencing of Intestinal DMT1 Mitigates Iron Loading in β-thalassemia Intermedia (Hbbth3/+) Mice

β-thalassemia is an iron-loading anemia caused by homozygous mutations in the hemoglobin subunit beta gene. In β-thalassemia major, frequent blood transfusions and elevated absorption of dietary iron can lead to iron overload, which exacerbates pathological outcomes. Iron absorption is inappropriately high due to blunted expression of the iron-regulatory hormone hepcidin. Low hepcidin potentiates iron export from duodenal enterocytes by ferroportin (FPN); cellular iron depletion then secondarily increases expression of the iron importer, divalent metal-ion transporter 1 (DMT1). High expression of both transporters drives excessive iron absorption and ultimately, iron overload. In non-transfusion-dependent, β-thalassemia intermedia (βTI), hepcidin suppression and high iron absorption are the sole causes of iron loading. In this investigation, two parallel hypotheses were tested in βTI ( Hbbth3/+) mice: 1) Intestinal DMT1 drives elevated iron absorption and precipitates iron overload; and 2) In vivo silencing of intestinal DMT1 mitigates iron loading. Initial experiments revealed that intestine-specific ablation of DMT1 caused a pathophysiological shift from iron overload to an iron-deficiency phenotype with exacerbated anemia, thus proving that intestinal DMT1 is required for iron absorption in Hbbth3/+ mice. Subsequent studies utilized folic acid-coupled, ginger nanoparticle-derived lipid vectors to deliver functional DMT1 siRNAs to the duodenal epithelium of 4-week-old Hbbth3/+ mice daily for 16 days to assess the impact on iron loading. Knockdown of intestinal DMT1 blunted iron loading in Hbbth3/+ mice, thus supporting hypothesis 2. These notable experimental outcomes establish intestinal DMT1 as a plausible therapeutic target to mitigate iron overload in βTI and possibly other non-transfusion dependent, iron-loading anemias. This work was supported by grants R01 DK074867 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK), and R01 DK109717 from NIDDK and the Offce of Dietary Supplements (to JFC). This is the full abstract presented at the American Physiology Summit 2024 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.

Ineffective matching of oxygen supply to myocardial metabolism in primiparous compared to nulliparous swine

Maternal mortality rates in the United States are higher than any other developed country in the world. Most postpartum maternal deaths are directly related to adverse cardiovascular events and often occur in mothers who have had a healthy pregnancy. Although pregnancy is recognized as a cardiovascular stress test for the pregnant patient, understanding of how the longitudinal and dynamic cardiovascular adaptations during pregnancy contribute to augmented risk of cardiovascular complications through the postpartum period remains poorly understood. We propose that pregnancy-induced adaptations in the coronary circulation contribute to adverse cardiovascular outcomes, especially during the puerperium period (1-60 days following delivery). Accordingly, this study tested the hypothesis that the balance between coronary blood flow and myocardial metabolism is compromised in the puerperium heart. Experiments were performed in anesthetized, open-chest control nulliparous (control) and primiparous (postpartum) swine during the puerperium period (35-45 days post-delivery). Systemic and coronary hemodynamic responses were assessed under baseline conditions and in response to continuous intravenous infusion of dobutamine (1 – 30 μg/kg/min). Under baseline conditions, blood pressure averaged 107 ± 6 mmHg in both control and postpartum swine and was unaffected by dobutamine administration (P = 0.94). Heart rate increased from 77 ± 6 to 165 ± 9 beats/min in response to dobutamine in control swine vs. 52 ± 3 to 139 ± 13 beats/min in postpartum swine (P = 0.53). Coronary blood flow and myocardial oxygen delivery were significantly diminished by ~60 ± 8% at rest in postpartum swine, which is consistent with reduced myocardial oxygen consumption (MVO2). The relationship between coronary blood flow and heart rate (P < 0.0001) or MVO2 (P = 0.0002) showed a marked parallel downward shift in postpartum swine at rest and in response to dobutamine infusion. This reduction in myocardial perfusion was associated with significant reductions in myocardial lactate uptake at rest in postpartum vs. control (0.34 μmol/min/g vs. 0.08 μmol/min/g, P = 0.02). Dobutamine increased myocardial lactate uptake in control by ~47 ± 3% with little change in the postpartum group. Together, these results suggest an ineffective matching of myocardial oxygen supply to metabolism in postpartum swine. This imbalance is consistent with increased incidence of myocardial ischemia and cardiac deaths in postpartum puerperium period. This work was supported by AHA.23PRE101281, NIH R01 HL158723. This is the full abstract presented at the American Physiology Summit 2024 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 Concurrent Administration of Dexamethasone and Aldosterone Effectively Mitigates the Pronounced Pro-ulcerogenic Effect of Dexamethasone on the Gastric Mucosa in Rats

We previously established that the effect of dexamethasone (Dex) on the gastric ulceration is time dependent. Administering Dex 1 hour before an ulcerogenic stimulus provides gastroprotection, but extending the interval to 24 hours transforms its effect into a pro-ulcerogenic one. The prolonged maintenance of blood glucose levels induced by Dex, accompanied by catabolic effects, may, in part, account for its pro-ulcerogenic nature (Filaretova et., Inflammopharmacology 2009). Given the evidence that Dex primarily binds to glucocorticoid receptors (GRs), we hypothesized that Dex-induced imbalance in the activation of GRs and mineralocorticoid receptors (MRs) could contribute to the pro-ulcerogenic effect of Dex. We partly confirmed this (Filaretova and Sudalina, FASEB J. 2019). To further validate this hypothesis, we conducted a study to determine whether correcting the imbalance in the activation of GRs and the MRs, induced by Dex, through co-administering Dex with the MR agonist aldosterone (Aldo), can mitigate the pro-ulcerogenic effect of Dex. The effects of separate and combined administration of Dex and Aldo at a dose of 1 mg/kg (i.p.) 1 hour and 24 hours before the onset of ulcerogenic stimulus on the gastric erosions was evaluated in rats. Gastric injury was induced by indomethacin (IM, 35 mg/kg, sc) in preliminary fasted rats. Four hours after IM administration, the rats were decapitated, the stomachs were removed to estimate the erosion area; the adrenal glands, thymus and spleen were remove to estimate their weight, and trunk blood was collected to test the corticosterone and glucose levels. We confirmed the gastroprotective effect of Dex administered 1 hour and the pronounced pro-ulcerogenic effect of Dex administered 24 hours before IM. Aldo itself, administered 1 hour before IM had no effect on the gastric erosions, however, when administered 24 hours before IM, it led to a pro-ulcerogenic effect of IM, but much less pronounced comparing with that of Dex. Co-administration Dex and Aldo 1 hour before IM resulted in the gastroprotection similar to that of Dex alone. Co-administration Dex and Aldo 24 hours before IM effectively mitigates the pronounced pro-ulcerogenic effect of dexamethasone on the gastric mucosa. Dex administration resulted in a sharp decrease in the blood corticosterone level both when administered 1 hour and 24 hours before IM, however, the corticosterone levels in 24 hours were significantly lower than in 1 hour. Aldo itself did not lead to a significant decrease the corticosterone levels neither after 1 hour nor after 24 hours. We confirmed our previous results that Dex administration both 1 hour and 24 hours before IM leads to an increase in blood glucose levels. Administration of Aldo alone did change glucose levels. The effect of the combined administration of Dex and Aldo on glucose levels did not differ from that of Dex alone. 24 hours after Dex administration alone or co-administration Dex with Aldo, pronounced catabolic effects appeared: a decrease in the relative weight of the adrenal glands, thymus and spleen. In conclusion, the concurrent administration of Dex and Aldo effectively mitigates the pronounced pro-ulcerogenic effect of Dex on the gastric mucosa in rats. The results further confirmed our hypothesis. State Program 47 SP “Scientific and Technological Development of the Russian Federation” (2019-2030), 0134-2019-0001. This is the full abstract presented at the American Physiology Summit 2024 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.

Availability of receptors for advanced glycation end-products (RAGE) influences differential transcriptome expression in mice exposed to chronic secondhand smoke

The receptor for advanced glycation end-products (RAGE) has a central function in orchestrating inflammatory responses in multiple disease states. RAGE is a transmembrane pattern recognition receptor with particular interest in lung disease due to its naturally abundant pulmonary expression. Our previous research demonstrated an inflammatory role for RAGE following acute exposure to secondhand smoke (SHS). However, chronic inflammatory mechanisms associated with RAGE remain unclear. In this study, we assessed transcriptional outcomes in mice exposed to chronic smoke in the context of RAGE expression. RAGE knockout (RKO) and wild type (WT) mice were exposed to SHS five times weekly via a nose-only delivery system (Scireq Scientific, Montreal, Canada) for six months and compared to mice exposed to room air (RA) only. Total lung RNA was isolated using the Direct-zol RNA MiniPrep kit (Zymo Research, Irvine, CA) and mRNA was purified using poly-T oligo-attached magnetic beads. Synthesis of cDNA, library construction, and sequencing was performed using standard approaches. We specifically compared the phenotypic and environmental conditions from WT+RA, WT+SHS, and RKO+SHS mice. Preprocessing and analysis of RNA-sequencing gene expression data included read trimming, mapping and quantifying the reads to transcripts, and calculating significant differentially expressed genes. The results of these analyses were summarized and compared via Venn diagrams, volcano plots, and functional gene cluster enrichment analysis. Notable gene clusters were specific to cytoskeletal elements, inflammatory signaling, and ciliogenesis. Finally, gene ontologies (GO) demonstrated significant biological pathways that were differentially impacted by the presence of RAGE. These data collectively identify several opportunities to further dissect RAGE signaling in the context of SHS exposure and foreshadow possible therapeutic modalities. This work was supported by funding from the National Institutes of Health (NIH 1R15-HL152257). This is the full abstract presented at the American Physiology Summit 2024 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.

Gold nanoparticles-conjugation of irisin enhances therapeutic effect by improving cardiac function and attenuating inflammation in sepsis

Introduction: Irisin is considered to be a promising therapeutic approach for cardiac depression and inflammatory disorders. The short half-life of irisin impeded its use and drug effcacy in the treatment. Objective: This study aimed to examine if pegylated gold nanoparticles-conjugated to irisin would improve therapeutic effects in cecal ligation and puncture (CLP)-induced sepsis in mice. Method: Recombinant irisin were conjugated to a pegylated gold nanoparticle, which was given to mice exposed to CLP. The cecal ligation procedure and sham on mice were operated and assigned to one of following five groups: I) CLP group: The mouse models underwent the CLP surgical procedure and received only vehicle saline treatment (n= 5); II) CLP + soluble Irisin: The mouse underwent the CLP and received an i.m (TA) injection of 1 ug of soluble irisin into each leg (n= 5); III) CLP + Gold nanoparticle-conjugated to Irisin: The mouse models underwent the CLP and received an i.m (TA) injection of 1 μg of Gold nanoparticle-irisin via intramuscular injection (TA) into each leg w (n= 5); IV) CLP + Gold nanoparticles- conjugated to IgG: The mouse underwent the CLP and received an i.m (TA) injection of gold nanoparticles conjugated to IgG (n=5). V) Sham: The mouse underwent the surgical operation without conducting the CLP (n=10). The post-operated animals were observed for one week, and survival rates were estimated. Echocardiography was performed to measure cardiac function at 12 hours following CLP. TUNEL was employed to detect apoptosis in both cardiac and skeletal muscles; histology was conducted to assess tissue injury in muscles. Enzyme linked immunosorbent assay (ELISA) was conducted to examine release of interleukin 6 (IL6) and the tumor necrosis factor (TNF) alpha. Results: Compared to the CLP control, soluble irisin treatment improved cardiac function recovery, as indicated by the fractional shortening (FS) and ejection fraction (EF). Irisin treatment exhibited reduced IL6 and TNF-alpha release in association with less apoptosis, lower muscle injury index and improved survival post-CLP. However, compared to soluble irisin treatment, gold nanoparticles-conjugated to irisin showed a significant improvement in cardiac function, suppression of apoptosis, reduced IL6 and TNF-alpha releases, decreased muscle injury and an improved survival rate of post-CLP. Conclusion: This study reveals that gold nanoparticles-conjugated irisin can serve to improve irisin’s therapeutic effects over a longer course of treatment. National Institute of General MedicalSciences (R01GM 141339); National Heart, Lung, andBlood Institute Grants (R01 HL089405). This is the full abstract presented at the American Physiology Summit 2024 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.

Aldo-keto reductase family 1 member A1 contributes to increase the glycolytic metabolism and to accelerate cellular senescence of bone derived mesenchymal stem cells

Skeletal aging characterizes the important event where bone loss is accompanied by accumulation of marrow adipose tissues. This event may be due to dysfunctional differentiation of bone derived mesenchymal stem cells (BMSCs) switched from osteoblasts (OBs) to adipocytes (ADs). Our accomplished work demonstrated that Aldo-keto reductase family 1 member A1 (Akr1A1) might serve as an important control to modify the NAD+-dependent SIRT signaling pathway for regulating the adipo-osteogenic lineage differentiation of BMSCs. In this study, we established a senescence cell model for examining whether increased Akr1A1 expression in BMSCs contributes to preferential lineage differentiation to ADs at the expense of osteogenesis by switching on the glycolytic metabolism in senescent cells with D-galactose induction or natural aging in MSCs. Our result suggests that increased expression of the glycolytic enzyme, Akr1A1 contributes to increase the glycolytic metabolism and to accelerate cellular senescence of MSCs. Such information might be useful to help develop the novel therapeutic strategy for treating age-related bone diseases with “perspective Akr1A1 inhibitors”. Grants supported by NSTC (112-2314-B-005 -004 -, NSC-111-2314-B-005-003-), iEGG and Animal Biotechnology Center from the Feature Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE-112-S-0023-A) in Taiwan, and partially by the grants of CYCH-NCHULS110003, CYCH-NCHULS111003, CYCH-NCHULS112002, TCVGH-NCHU 112-7616 to Y-ML. This is the full abstract presented at the American Physiology Summit 2024 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.

Science Communication Training for Graduate Exercise Physiology Students

Physiologists have a responsibility to inform the public about how new discoveries and challenges will impact the health of society. However, most physiologists do not have formal training in science communication. The purpose of this study was to implement a science communication training pilot program for graduate students. Students (n = 23) were introduced to science communication training that emphasized key skill development (writing, public speaking, leadership, project management, teaching) as part of their advanced exercise physiology course. Subsequently, students carried out service-learning projects that tasked them with presenting evidence-based health information to public audiences (e.g., community members, clinicians, K-12 students, journalists, policy makers). In a subset of students (n = 8), a survey was administered to assess students’ perceptions of the value of science communication and their ability to communicate to different audiences. Service-learning projects that students completed included blog posts, infographics, podcast series, visits with K-12 schools, and public townhall presentations. Most students (≥75%) agreed that completing the science communication training helped them develop confidence speaking to a range of audiences. After completing the science communication training, students were more interested in learning about how to better communicate science concepts to the public compared to before the training (88% vs. 25%). Collectively, these preliminary results suggest that science communication training helped graduate exercise physiology students recognize the gap between science and society, develop confidence speaking to a range of audiences, and communicate more effectively. These results support findings from other disciplines such as biology and medicine. Implementation of science communication training is paramount, and the approach described here could be useful for physiology students, educators, and departments. American Physiological Society - Teaching Career Enhancement Award. This is the full abstract presented at the American Physiology Summit 2024 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.

Multi-Functional Role of RECK in Metabolic Dysfunction-Associated Steatotic Liver Disease Development: A Mass Spectrometry-Proteomic Approach

Metabolic dysfunction-associated steatotic liver disease (MASLD, previously referred to as nonalcoholic fatty liver disease) is characterized by increased fat deposition and inflammation in the liver as well as dysregulation of cellular processes that affect the extracellular matrix (ECM). We previously observed that the expression of Reversion Inducing Cysteine Rich Protein with Kazal motifs (RECK), a key ECM regulatory protein, negatively correlates with MASLD-associated fibrosis and inflammation (metabolic dysfunction-associated steatohepatitis, MASH). However, mechanisms governing this relationship remain unclear. Therefore, we hypothesized that overexpression of RECK is hepatoprotective in the setting of MASH by altering several ECM-linked pathways, as analyzed by proteomics. Accordingly, we generated hepatocyte-specific Reck-overexpressing transgenic mice. Male hepatocyte-specific Reck-overexpressing transgenic mice and littermate controls were fed a Western diet high in fat, carbohydrate, and cholesterol for 24 weeks (n=6/group). Whole liver tissue was submitted for mass spectrometry-proteomic analysis. We identified 370 differentially expressed proteins out of a total of 4693 proteins using a q-value cutoff of ≤0.10; of which 90 proteins were significantly upregulated while 280 significantly downregulated. This included an upregulation of Cyp2b (adjusted p≤0.01) and downregulation of Taok1, C/EBPβ, and Vitronectin (all adjusted p≤0.01), all of which have been shown to be important in liver health and metabolism in the context of MASH. Predictive pathway analysis using Ingenuity Pathway Analysis software (Qiagen) identified several significantly altered pathways, including those involved in ECM regulator pathways (integrin signaling, z-score=6.0; mTOR signaling, z-score=5.3; leukocyte extravasation signaling, z-score=5.1), cell-signaling (chemokine signaling, z-score=5.4; CXCR4 signaling chemokine signaling, z-score=5.0), and inflammatory responses (leukocyte extravasation signaling, z-score=5.1; CXCR4 signaling, z-score=5.0). Interestingly, pathway analysis based on significantly altered proteins predicted the downregulation of critical components of hepatic inflammation and fibrosis, including TGF-β, IL-1β, and TNFα. These novel results reveal the regulatory role of RECK in MASH progression, specifically governing the components of ECM remodeling and inflammation. Our goal is to develop RECK inducers and identify additional therapeutic targets in MASH development and progression. This work was funded by the NIH award R01DK130243. This is the full abstract presented at the American Physiology Summit 2024 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.

EphB2 deficiency in hepatic stellate cells mitigates fibrogenesis in metabolic dysfunction-associated steatohepatitis by attenuating the TGFβ/SMAD signaling

Background & Aims: The receptor tyrosine kinase EphB2 is now emerging as a critical regulator of inflammation and fibrogenesis in metabolic dysfunction-associated steatohepatitis (MASH) though the underlying mechanism remain unclear. We aimed to investigate the role or mechanism by which EphB2 contributes in the progression of MASH fibrogenesis. Hypothesis: We hypothesized that EphB2 in hepatic stellate cells (HSCs) promotes MASH-liver fibrosis Methods: Single nuclear RNA-sequencing of liver from human and mouse MASH were analyzed for EphB2 expression. Human liver biopsies obtained from patients with MASH were used to assess EphB2 expression by immunohistochemistry. Primary human HSCs were stimulated with TGFβ with or without TGFβR/SMAD inhibitors (SB525334, CS208, and SIS3 at 1μM) and EphB2 expression assessed by qPCR, western blot and immunofluorescence staining. EphB2 was silenced in primary human HSCs using siRNA and the resulting HSCs assess for TGBβ-induced HSC-myofibroblasts transition. MASH models were developed in hepatocyte-specific EphB2 knockout and HSCs EphB2 knockout and wild-type mice fed a high fat diet for 26 weeks or a choline-deficient amino-acid defined (CDAA) high fat diet for 10 weeks. Statistical analysis was performed using GraphPad Prism and a p-value < 0.05 was considered significant. Results: The expression of EphB2 was 3-fold significantly upregulated in liver specimens from patients with advanced MASH and correlated with disease severity. EphB2 was modestly expressed in hepatocyte, but strongly induced in HSCs in human and mouse MASH. Hepatocyte-specific EphB2 deficiency does not inhibit the development of steatohepatitis and fibrosis in mice fed the CDAA-diet. TGFβ/SMAD signaling promotes an 8-fold increase of EphB2 expression in human HSCs while siRNA silencing of EphB2 in human HSCs was suffcient to abolished TGFβ-mediated HSCs activation. Interestingly, HSCs-specific EphB2 knockout mice developed 2.5 to 6-fold less fibrosis than wild-type controls in response to high fat diets feeding suggesting that EphB2 expression on HSCs is required for the development of fibrogenesis in MASH. Deletion of EphB2 in activated HSCs after fibrosis was established in mice was suffcient to mitigate MASH fibrosis. Conclusions: EphB2 is upregulated in human and mouse MASH. Upregulation of EphB2 in human HSCs is mediated via the TGFβ/SMAD signaling. HSCs expressing EphB2 regulate the development of MASH-fibrosis. Specific EphB2 inhibitors could potentially mitigates fibrogenesis in MASH, opening up an avenue for the treatment of MASH. Funding sources: NIDDK, NIAMS, APS postdoctoral fellowship. This is the full abstract presented at the American Physiology Summit 2024 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.

Enteric and Systemic Postprandial Lactate Shuttle Phases and Dietary Carbohydrate Carbon Flow

We interrogated the hypothesis of a Postprandial Lactate Shuttle (PLS) in young, healthy men and women (n=15). Participants reported to the lab (5:00 AM) following an overnight fast and had the forearm vein catheterized for primed continuous infusions of [6,6-2H]glucose and [3-13C]lactate stable isotope tracers. The contralateral hand vein was warmed and catharized for arterialized blood sampling. After a 90-minute (min) equilibration period participants underwent a 75 g oral glucose tolerance test (OGTT). Arterialized [lactate] rose from baseline (0.60sup>). We estimate carbon flow from the OGTT from the rates of appearance of glucose, lactate and GNG from lactate. At 120-min the 75 g load was accounted for as follows: 29 g as blood lactate, 24 g of glucose from hepatic glucose release, 8 g of glucose from GNG, and 14 g withheld in the liver. Because blood [lactate] and Ra rose before [glucose] and Ra, evidence of an Enteric PLS is provided. Because secondary increments in blood [lactate] and Ra coincided with those of glucose, results are interpreted to indicate presence of a larger, secondary Systemic PLS phase. Antiquated ideas of lactate production due to hypoxia in skeletal muscles need to be supplanted because glycolysis proceeds to lactate in fully aerobic tissues, and because dietary carbohydrate is processed via lactate shuttling. In humans, lactate is a major vehicle for carbohydrate carbon distribution and metabolis. NIH grant R01 AG059715. This is the full abstract presented at the American Physiology Summit 2024 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 impact of increased daily peanut consumption on indices of peripheral and cerebral vascular function in non-Hispanic Black individuals

Cardiovascular disease, neurocognitive conditions, and cerebral vascular diseases including cognitive impairment, stroke, and Alzheimer’s disease and related dementias disproportionately impact the Non-Hispanic Black (BL) population. The mechanisms are multifactorial but is related to reduced vascular function/health. Our lab, and others, have demonstrated that reduced nitric oxide (NO) bioavailability secondary to oxidative stress, systemic inflammation, and reduced L-arginine bioavailability contributes to reduced vascular function/health in this population. Increased dietary peanut consumption improves various physiological outcomes including blood pressure, cholesterol, type II diabetes, cognitive health, NO bioavailability, and vascular function/health. This improvement is related to increased bioavailability of L-arginine, reduced systemic inflammation, and antioxidant capacity. This study hypothesized that increased daily peanut consumption would improve indices of peripheral and cerebral vascular function/health in a cohort of BL individuals. Methods: Seventeen people participated (11 non-Hispanic White (WH) and 6 BL (age: WH 25±8, BL 21±2; BMI: WH 23±3, BL 22 ± 4; p>0.05 for both). Measures were made before (pre) and after (post) 56 days of daily lightly salted peanut consumption (2 oz/day for females and 3 oz/day for males). Peripheral macro and microvascular function/health was assessed as brachial artery vasodilation (flow mediated dilation, %FMD) and forearm reactive hyperemia (peak mean forearm blood velocity (FBVmean) respectively following 5-min of suprasystolic cuff inflation. Cerebral vascular function/health was assessed as the cerebral vasodilator response during a hypercapnic challenge, i.e., the slope of % cerebral vascular conductance index (CVCi) relative to the change in end-tidal carbon dioxide concentration (ΔPETCO2). Results: There was no effect of race on peripheral macrovascular function assessed as %FMD (WH: 5.3 ±0.89, BL: 6.3 ±1.9 %, p=0.31) or peripheral microvascular function assessed as FBVmean (WH: 82.90 ± 4.5, BL: 72 ± 6.7 cm·s−1, p=0.13). Likewise, there no effect of treatment on %FMD (Pre: 5.4 ± 0.7, Post: 6.2 ± 2.1 %, p=0.46) or FBVmean (Pre: 81.4 ± 6.7, Post: 73.4 ± 8.8 cm·s−1, p=0.27). Lastly, there were no effect of race (WH: 4.0 ± 0.36, BL: 3.9 ± 0.6 %, p=0.85) or treatment (Pre: 3.9 ± 0.56, Post: 4.0 ± 0.39 %, p=0.79) on cerebral vascular function assessed as %CVCi during hypercapnia. Conclusion: The preliminary data suggest that 56 days of increased daily peanut consumption does not induce measurable improvements in indices of vascular function in a cohort of relatively young BL individuals. Future work will continue to assess this effect in a larger cohort of individuals as well as in individuals with existing conditions or diseases (i.e., hypertension, type II diabetes, etc.). Funding: Peanut Institute (982307). This is the full abstract presented at the American Physiology Summit 2024 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.

Mechanosensor channels of the lens and ciliary body: patch clamp studies

Epithelia from the lens and the ciliary body possess mechanosensors, including Piezo1 and transient receptor potential (TRP) channels, that may partake in the regulation of lens and aqueous humor volume. Although these mechanosensor channels commonly allow calcium entry, the outcomes of elevated intracellular calcium vary with the type of channel activated. For instance, separate activation of TRPV4 and TRPV1 increases Na-K-ATPase and Na/K/2Cl cotransporter activities, respectively. To further understand the role of mechanosensitivity in lens and ciliary body, we are currently using patch clamp techniques to characterize the response of primary cultured cells to chemical agonists and antagonists, as well as direct mechanical stimulation. Previously, we found that in mouse lens epithelial cells (mLEC), TRPV4 activation by agonist GSK1016790A (GSK) is followed by disappearance of a fast, transient K+-like outward current (Ipeak), decrease of a steady state outward current (Iss), fast depolarization of resting membrane potential (RMP) values from near -50 mV to near 0 mV, increase of Cx43 hemichannel-like events, and increase of overall membrane conductance ( gm) at holding potential (HP) = -50mV. More recently, we found that in mLEC, shear stress (streaming of external solution from a pipette tip over the cell membrane) reduced Ipeak but did not cause large RMP changes. Porcine non-pigmented epithelial cells (pNPE) in normal conditions display no Ipeak, small Iss, small RMP near -20 mV and spontaneous Cx43 hemichannel-like event activity. Nevertheless, GSK caused further depolarization of pNPE cells, and while no gm changes were seen when GSK was applied while holding cells at -50 mV, gm did increase when GSK exposure occurred during repeated ±80 mV pulses. Preliminary experiments show that activation of TRPM3 by its agonist pregnenolone causes a minimal hyperpolarization in mLEC. In comparison, in a line of human LEC (HLE B3) expressing TRPM3 channels, and which appear generally depolarized, pregnenolone caused a proportionally larger shift toward more negative RMP values; more intriguing, GSK has no measurable effect on the gm of HLE B3 cells. Taken together, and apart from considerations of cell- and species-specific differences, the data suggest that mechanosensor channels have different and/or opposite effects on the regulation of membrane ion channel activity, and thus on cell function. R01EY029171 and R01EY009532. This is the full abstract presented at the American Physiology Summit 2024 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.

Role of Protein Phosphatase 1 (PP1) Upon KCCs Regulation by WNK3 Kinase During Cell Volume Regulation and Ion Transport

Background: The K-Cl cotransporters (KCCs) are SLC12A cation/chloride cotransporters (CCCs) that mediate epithelial transport, maintain cellular volume, and regulate GABA neurotransmission. These mechanisms are complex, and contain multiple sensors, transducers, and effectors, often operating in parallel pathways. It has been shown that KCCs activity is affected by direct phosphorylation and dephosphorylation. In particular dephosphorylation of important regulatory sites on KCCs is necessary to become fully activated, whereas phosphorylation by SPAK/OSR1 kinases will result in KCCs inhibition. Protein phosphatases (PP) inhibitors reduce KCCs function and so, it is generally accepted the presence of phosphatases on the KCCs signaling pathway. Several studies have proposed protein phosphatase 1 (PP1) as the major PP responsible for KCCs dephosphorylation. On the other hand, WNK3 kinases have been shown to directly regulate KCCs activity bypassing their osmotic regulation. That is wild type (WT) WNK3 phosphorylates KCCs under hypotonic conditions where they are normally active, whereas kinase dead WNK3 (DA) activates KCCs function under isotonic conditions where they are normally inhibited. On the present study we analyze the role of protein phosphatase 1 (PP1) upon KCCs regulation by WNK3 kinase during cell volume regulation and ion transport. Methods: HEK 293 cells and Xenopus laevis oocytes were either transfected or microinjected with KCC3 or KCC4 and one of the following PP1 plasmids: WT, H248K (inactive) or T320A (constitutive active), and/or WT WNK3 /DA WNK3. KCCs dephosphorylation in response to cell swelling, hypotonic conditions, was evaluated by Western blot analysis of KCCs regulatory phosphorylation site (pThr1048 in KCC3a). Phosphatase/kinase interaction and the possible formation of a complex was analyzed by transfecting cells or microinjecting oocytes with WNK3 PP1 putative binding sites mutants (WNK3 PP1a y PP1b). Phosphorylation of SPAK/OSR1 activating regulatory site Ser373 was analyzed in parallel under the same conditions. Results: WT or constitutive active mutant PP1 T320A induced KCCs Thr1048 dephosphorylation in HEK cells and X. laevis oocytes even under isotonic conditions, where KCCs are normally phosphorylated. WT WNK3 prevented KCCs T1048 dephosphorylation under hypotonic conditions, ablating their response to cell swelling. Nevertheless, when co-expressed with PP1 T320A, KCCs became dephosphorylated in both osmotic conditions eliminating WT WNK3 inhibitory effect. PP1 inactivating mutation H248K prevented KCCs Thr1048 dephosphorylation in response to hypotonic conditions. However, when co-expressed with DA WNK3, this KCCs regulatory site, became dephosphorylated under hypotonic conditions. SPAK Ser373 regulation was unaffected in any off the studied conditions. Conclusions: Our results show a direct role for PP1 on KCCs function. PP1WT and PP1T320A regulate KCCs activity by its dephosphorylation. In presence of WT WNK3, PP1 activity predominates over WNK3 surpassing the inhibitory phosphorylation of the regulatory Thr residue. KCCs are not dephosphorylated by PP1H248K under hypotonic conditions. Coexpression of kinase dead WNK3 overcomes this effect, supporting the hypothesis of a phosphatase/kinase complex regulating KCCs cell volume regulatory response. AM CF-2023-I-532, INCar 22-1345 PdlH Papiit IN228123. This is the full abstract presented at the American Physiology Summit 2024 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.

Metabolic dysfunction in the myocardium three months after exertional heat stroke in mice

In humans, exposure to heat stroke has proven to be a risk factor for development of cardiovascular disease later in life. We hypothesized that exposure to exertional heat stroke (EHS) would result in the development of similar long-term cardiac disease in mice. In this study we followed mice for three months after EHS, using ultrasound imaging of the heart, metabolomics and histological analysis of ventricular tissue and biomarker analysis after 4 weeks of recovery. Methods: Mice were instrumented with temperature telemetry devices and were trained to run on forced running wheels using an incremental exercise protocol. EHS mice ran in an temperature elevated environmental chamber; females 37.5°C; males 34°C; whereas, matched exercise control mice (EXC) ran in 22-23°C. Running stopped when EHS mice reached symptom limitation (unconsciousness). The mice were followed for 12 wks post EHS, with two ultrasound measures at 2 and 10 wks of recovery. At the end of the study, mice were anesthetized, a blood sample drawn, and ventricular tissue collected for metabolomics. RESULTS: Male but not female EHS mice gained ~10% greater body mass compared to EXC over the course of recovery ( P<0.01). Hearts from male but not female EHS mice exhibited elevations in heart mass per tibia length ( P<0.01). At three months, metabolomics in male EHS hearts revealed significant elevations in glucose and glucose metabolites and significant decreases in acylcarnitines consistent with a metabolic shift toward glucose and away from lipid metabolism. In contrast, female hearts exhibited fewer abnormalities but with significant elevations in lipid and arachidonate metabolites. At two weeks post, there were no differences in male ultrasound measures between EHS and EXC mice but in females there was a reduced posterior wall velocity ( P=0.012) and a trend towards narrower posterior wall thickness ( P= 0.06) . Between 2 and 12 wks post EHS, there were significant elevations in fractional shortening in males ( P<0.05) and elevations in posterior wall velocity ( P<0.01). In contrast, females exhibited significant reductions posterior wall velocity. From blood samples, male EHS mice had increased HDL and females exhibited decreased HDL (p<0.01 in both). LDL increased only in males P<0.05. Vascular adhesion molecules sICAM ( P<0.01) and Pecam1 ( P<0.01) were significantly decreased in EHS males and females and e-Selectin was decreased in EHS males only ( P<0.001). Total serum protein was significantly reduced in both male and females ( P<0.01). Male EHS mice also exhibited significant elevations in white blood cell count and total lymphocytes ( P=0.01) suggesting ongoing activation of the immune system . CONCLUSIONS: In this controlled preclinical model of EHS, both male and female mice exhibited long-lasting cardiovascular and systemic disorders that could account for the lingering incidence of cardiovascular disease and poor overall health in heat stroke victims. Supported by US Army Med. Research and Devel. Command BA180078 and the BK and Betty Stevens Endowment. This is the full abstract presented at the American Physiology Summit 2024 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.

EGFR inhibition with erlotinib induces membrane accumulation of aquaporin-2 via PDK1 and RSK activation

In the canonical aquaporin-2 (AQP2) signaling pathway, vasopressin (VP) binds to its V2 receptor, which in turn activates adenylate cyclase and protein kinase A (PKA), resulting in AQP2 S256 phosphorylation and accumulation of AQP2 in the membrane. The epidermal growth factor receptor (EGFR) inhibitor erlotinib also increases AQP2 S256 phosphorylation and membrane accumulation, yet it does not increase cAMP or PKA activity. We hypothesize that the ribosomal s6 kinase (RSK), a downstream effector in the EGFR-MAPK/ERK pathway, is the terminal kinase phosphorylating AQP2 in this novel PKA-independent pathway activated by erlotinib, and that the phosphoinositide dependent kinase-1 (PDK1), a well-established activator of RSK, is indispensable for erlotinib-induced AQP2 activation. Using our LLC-AQP2 cell model, we show that erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation are abolished by the specific RSK inhibitor BI-D1870. RSK knockdown with siRNA also blocked AQP2 S256 phosphorylation and membrane accumulation, as did RSK knockout with CRISPR/cas9 editing. Next, rat kidney slices were incubated in Hank’s buffer with or without BI-D1870 and treated with erlotinib. BI-D1870 inhibited AQP2 membrane accumulation in collecting duct principal cells, supporting the relevance of this pathway in kidney cells in situ. An in-vitro kinase assay using purified proteins demonstrated that RSK directly phosphorylates AQP2 at the S256 residue. Additionally, erlotinib consistently increased phosphorylation of RSK T359, a residue associated with RSK’s active state. In canonical RSK activation, a phosphorylation cascade results in PDK1 binding to and activating RSK. We found that PDK1 inhibition with an experimental compound PS-222 also blocked erlotinib-induced AQP2 membrane accumulation and S256 phosphorylation. This implicates PDK1 along with RSK in the novel erlotinib-induced pathway of AQP2 traffcking. Our data show that RSK and PDK1 are terminal effectors in the novel, PKA-independent pathway of AQP2 activation induced by erlotinib. Further elaboration of this new non-canonical pathway promises to uncover potential pharmacological targets for the treatment of water balance disorders. This work was supported by the National Institutes of Health (NIH) grant DK096586 (D. Brown). P. W. Cheung was supported was supported by NIH K-award DK115901. Richard Babicz is the recipient of the Ben J Lipps Research Fellowship (American Society of Nephrology). Additional support for the Program in Membrane Biology Microscopy Core came from the Boston Area Diabetes and Endocrinology Research Center (DK057521) and the Massachusetts General Hospital (MGH) Center for the Study of Inflammatory Bowel Disease (DK043351). This is the full abstract presented at the American Physiology Summit 2024 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.

Exploring Serotonergic mechanisms in Opioid Induced Respiratory Depression

Opioids profoundly depress breathing which can be fatal in the case of overdose. Despite growing knowledge of the pontine and medullary respiratory control circuits that are inhibited by opioids, the circuit and cellular mechanisms of opioid induced respiratory depression are not fully understood. Serotonin neurons located in the caudal medullary raphe modulate breathing and possess mu opioid receptors. Therefore, we hypothesized that serotonin-producing raphe neurons contribute to opioid induced respiratory depression. To test this, we employed to experimental approaches: 1) arterially perfused in situ working heart brainstem preparations and 2) ex vivo acute brain slice electrophysiology to identify and characterize serotonin neuron inputs onto brainstem respiratory neurons. To specifically activate serotonin neurons, we used ePet-Cre::Ai32 mice which Cre-dependently express channelrhodopsin-2, a light-activated cation channel, in serotonin neurons. At the circuit level, using the in situ preparation, we found that activation of caudal medullary raphe serotonin neurons increased respiratory rate by decreasing inspiratory and expiratory duration. Fentanyl application blocked the respiratory effects induced by serotonin neuron activation which were subsequently restored using naloxone. These results suggest serotonin neurons in the caudal medullary raphe are inhibited by opioids. To characterize the serotonin-mediated mechanisms at the cellular level, we made acute brain slices containing dorsal raphe neurons and lateral parabrachial neurons from ePet-Cre::Ai32 mice. We verified that optical stimulation resulted in action potentials and serotonin release from dorsal raphe serotonin neurons. Optically evoked serotonin release caused 5HT1A receptor-mediated outward currents in dorsal raphe serotonin neurons. To locate and characterize serotonin neuron inputs onto lateral parabrachial neurons, we performed whole-cell voltage clamp recordings from lateral parabrachial neurons and delivered paired light pulses (10 ms duration, 50 ms interval). We observed optically evoked inhibitory post-synaptic currents (IPSCs) that were blocked by the GABA-A receptor antagonist gabazine (1 μM). Optically evoked IPSCs had long latency (> 5 ms) and high jitter (> 1 ms), indicating stimulation of serotonin terminals resulted in polysynaptic GABA release onto lateral parabrachial neurons. Future work will examine the influence of opioids on this raphe-parabrachial circuit. Uncovering the role of serotonin neurons in opioid induced respiratory depression may reveal targets for restoring breathing in the presence of opioids or other conditions where life-threatening apneas occur. This work was supported by the National Institutes of Health (NIDA) R01DA047978-05 grant to ESL. This is the full abstract presented at the American Physiology Summit 2024 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.

Divergent Effects of IL-18- and IL-18R-deficiency on Angiotensin II-induced Abdominal Aortic Aneurysm Formation

Currently, there are no pharmacological treatments for abdominal aortic aneurysms (AAA), and AAA rupture is fatal in ~90% of patients. Aortic inflammation is strongly correlated with the expansion of AAA and recently the NLRP3 inflammasome has been implicated in thedevelopment of AAA. Interleukin (IL)-18, an inflammasome-derived cytokine, exerts pro-inflammatory responses when bound to its cognate IL-18 receptor (IL-18Rα). The current project aimed to determine the effect of IL-18- and IL-18Rα-deficiency on the incidence of angiotensin (Ang) II-induced AAA. Ten-week-old male C57BL/6 (WT), IL-18-deficient ( Il18−/−) and IL-18Rα-deficient ( Il18r1−/−) mice were infused with Ang II (1.44 mg/kg/day, s.c.) for 28 days via osmotic minipump. Ang II-induced AAA incidence differed in all strains. While 52% of WT mice developed AAA (including premature deaths due to rupture), no AAAs were detected in Il18−/− mice (p<0.05, n=20-23). Strikingly, Il18r1−/− mice exhibited a higher incidence of AAA and associated mortality compared to WT mice(88%; p<0.05, n=23-25). Consistent with this finding, in vivo ultrasound imaging confirmed advanced dilation of the abdominal aorta at d7 after Ang II-treatment in Il18r1−/− mice cf. WT mice (1.49 ± 0.13 vs 1.15 ± 0.05 mm; p<0.05; n=23-25). Single cell RNA-sequencing of Ang II-infused mouse aorta revealed Il18r1 gene expression was limited to gamma-delta T cells, suggesting these cells may be driving IL-18 mediated AAA formation. In summary, IL-18-deficiency affords protection from AAA formation, while AAA incidence and severity were augmented in Il18r1−/− mice. Neutralisation of IL-18, but not IL-18Rα inhibition, may represent a novel therapeutic strategy to treat AAA. NHMRC (GNT1143674). This is the full abstract presented at the American Physiology Summit 2024 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.