bluuebunny/crossref_metadata_2025 huggingface dataset:
Neonatal Enteric Glia Enhance Intestinal Epithelial Restitution in vitro Following Exposure to Sterile Colonic Luminal Content of Mature but not Neonatal Pigs
Authors: Caldwell Madison , Van Landeghem Laurianne , Blikslager Anthony , Ziegler Amanda
Published: May 2023

Neonates exhibit significantly poorer outcomes from damage to the intestinal epithelium following ischemia for unknown reasons. In our pig model, we recently discovered an age-dependent epithelial restitution defect in ischemia-injured neonatal intestine that is rescued by application of homogenized juvenile mucosa, but the mechanism remains unknown. Enteric glial cells (EGC) have been shown to promote epithelial restitution following injury via paracrine signaling and are abundant in the intestinal mucosa of juvenile but not neonatal pigs. Co-culture studies have shown that pig EGC enhance restitution in epithelial monolayers following scratch wounding. In mice, postnatal maturation and maintenance of the EGC network is purportedly driven by colonization of gut microbiota. Furthermore, weaning of neonates leads to drastic shifts in the dynamics and populations of these gut microbiota. Therefore, we believe that changes to pig intestinal microbiota during weaning may play a key role in EGC maturation required for proper epithelial restitution. We hypothesized that treatment with mature (>6 weeks of age), but not neonatal (2 weeks of age), luminal content would improve restitution of neonatal epithelial monolayers co-cultured with neonatal EGC. We compared the effect of sterile-filtered neonatal or mature luminal content addition to the apical chamber on scratch wound restitution in neonatal porcine IPEC-J2 monolayers in monoculture or co-culture with primary porcine neonatal submucosal EGC using a transwell system (n = 9). Our results support a significant effect of luminal content treatment in the presence of EGC (P=0.0085). Specifically, mature luminal content treatment enhanced IPEC-J2 wound closure in co-culture with EGC but not in IPEC-J2 monoculture (P=0.0156). EGC co-culture alone or treated with neonatal lumen content did not affect restitution. Both neonatal and mature luminal content treatment decreased restitution in monoculture, but this effect was not significant. These data suggest that EGC provide secretory signals to the intestinal epithelium that promote repair in response to a component of mature luminal content, which we believe to be microbial products. Future work aims to determine what is driving these differences in restitution by analyzing neonatal and mature luminal secretomes.

NIH 5 T35 OD 11070-12; NIH K01 OD 028207; NIH P30 DK 034987; NIH-NICHD R01 HD095876; USDA-NIFA VMCG-0065.

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.
Interleukin-12 alpha deficiency attenuates pressure overload-induced cardiac inflammation, hypertrophy, dysfunction, and heart failure progression
Authors: Bhattarai Umesh , He Xiaochen , Xu Rui , Liu Xiaoguang , Pan Lihong , Sun Yuxiang , Chen Jian-Xiong , Chen Yingjie
Published: May 2023

IL-12α plays an important role in modulating inflammatory response, fibroblast proliferation and angiogenesis through modulating macrophage polarization or T cell function, but its effect on cardiorespiratory fitness is not clear. Here, we studied the effect of IL-12α on cardiac inflammation, hypertrophy, dysfunction, and lung remodeling in IL-12α gene knockout (KO) mice in response to chronic pressure-overload produced by transverse aortic constriction (TAC). Our results showed that IL-12α KO significantly ameliorated TAC-induced left ventricular (LV) failure, as evidenced by a smaller decrease of LV ejection fraction and fractional shortening. IL-12α KO also significantly attenuated TAC-induced increase of LV weight, left atrial weight, lung weight, right ventricular weight, and their ratios to body weight or tibial length. In addition, IL-12α KO significantly attenuated TAC-induced LV leukocyte infiltration, fibrosis, cardiomyocyte hypertrophy, and lung inflammation and remodeling (such as lung fibrosis and vessel muscularization). Moreover, IL-12α KO significantly attenuated TAC-induced activation of CD4+ T cells and CD8+ T cells in the lung. Furthermore, IL-12α KO significantly suppressed accumulation and activation of pulmonary macrophages and dendritic cells. Taken together, these findings indicate that inhibition of IL-12α is effective in attenuating pressure overload-induced cardiac inflammation, heart failure development, promoting transition from LV failure to lung remodeling and right ventricular hypertrophy.

This work was supported by research grants R01HL161085, R01HL139797, P20GM104357, and P01HL51971 from NIH.

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 regulatory role of Sirtuin 6 in coronary endothelial dysfunction in HFpEF
Authors: Wang Yang , Enrick Molly , Gadd James , Yin Liya
Published: May 2023

Sirtuin 6 (Sirt6) has been implicated in obesity, insulin resistance, type 2 diabetes mellitus, and cardiovascular diseases. Sirt6 protects EC from premature senescence, oxidative stress, and atherosclerosis by sustaining high eNOS levels and preserving cell replication. A recent publication showed that overexpressed Sirt 6 in EC mitigated heart failure with preserved ejection fraction (HFpEF) in diabetes. We hypothesize Sirt 6 regulates coronary microvascular dysfunction (CMD) in HFpEF associated with diabetic cardiomyopathy. CMD is characterized by impaired endothelial-dependent vasodilation, but detailed mechanisms have yet to be elucidated. How Sirt6 regulates coronary microvascular function remains to be determined. Inducible Sirt6 endothelial-specific knockout and wild-type (WT) mice were fed a chow or high fat and sugar (HFHS) diet. Coronary arterioles were isolated, and endothelial-dependent vasodilation (EDD) was assessed using myography (DMT). Echocardiography and treadmill exercise exertion tests were performed to evaluate cardiac function. Myocardial blood flow (MBF) was measured by doppler. Cardiac fibrosis was detected using trichrome staining, and molecular pathways were elucidated via gene and protein analysis. Our preliminary data show that the EDD of coronary arterioles of Sirt6 endothelial-specific knockout treated with HFHS was impaired, and the mediator of coronary vasodilation switched from NO to H2O2 in the Sirt6 knockout mice. Compared to the WT mice, myocardial blood flow and cardiac function were changed in Sirt6 knockout mice. Our data suggest that Sirt6 regulated coronary microvascular function through endothelial cells, and ablation of Sirt6 in endothelial cells caused CMD and diastolic dysfunction, eventually HFpEF. Further genetic profiling will elucidate the pathways and mechanisms converging with Sirt6 to regulate microvascular function in HFpEF.

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.
Rhabdomyolysis-induced renal ion accumulation promotes early acute kidney injury in sickle cell mice
Authors: Williams Jada , Kumar Ravi , Afolabi Jeremiah , Park Frank , Adebiyi Adebowale
Published: May 2023

Individuals with sickle cell disease (SCD) are at greater risk for developing rhabdomyolysis, a potentially life-threatening syndrome resulting from the breakdown of skeletal muscle fibers. Acute kidney injury (AKI) is one of the most severe complications of rhabdomyolysis. Chronic kidney and cardiovascular disease, which account for SCD mortality, are long-term consequences of AKI. Although SCD elevates the risks of rhabdomyolysis-induced sudden death, the mechanisms that underlie rhabdomyolysis-induced AKI in SCD are unclear. Here, we examined the effects of glycerol-induced rhabdomyolysis on kidney health in transgenic humanized mice that express human sickle hemoglobin (HbSS-Townes model). Unlike the control mice (AA), homozygous SCD mice (SS) exhibited 100% mortality 8-24 h after intramuscular glycerol injection. Five hours of glycerol-induced rhabdomyolysis caused a more significant increase in myoglobinuria and plasma creatine kinase levels in SS compared to AA mice. Since SS mice experience chronic hemolysis, releasing free heme and iron into the bloodstream, and the induction of rhabdomyolysis releases myoglobin, we examined heme and renal iron parameters in these mice. Basal plasma heme, iron accumulation, and kidney tissue ferric iron levels were significantly higher in SS than in AA control mice. In contrast to AA, rhabdomyolysis aggravated these parameters in SS mice. Rhabdomyolysis also amplified renal oxidative stress in SS compared to AA mice. SS mice exhibited worse renal function, exemplified by a significant decrease in GFR, increased plasma and urinary biomarkers of early AKI, and more significant renal tubular injury. The free radical scavenger TEMPOL ameliorated rhabdomyolysis-induced AKI in the SS mice. These findings suggest that rhabdomyolysis and subsequent AKI are heightened in SCD mice. It can also be inferred that oxidative stress driven by renal iron accumulation may underlie rhabdomyolysis-induced early AKI in SCD.

NHLBI: R01 HL151735-01 and R01HL151735-S

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.
PTSD induced inflammation negatively impacts cardiac homeostasis
Authors: Corker Alexa , Troncoso Miguel , Broughton Philip , Sidles Sara , Kelly Ryan , LaRue Amanda , DeLeon-Pennell Kristine
Published: May 2023

Multiple studies indicate that post-traumatic stress disorder (PTSD) is a risk factor for cardiovascular disease (CVD), however the mechanisms behind this correlation is unknown. We hypothesize that PTSD stimulates recruitment of monocyte-derived macrophages to the heart, resulting in increased cardiac fibrosis and resetting cardiac homeostasis. To induce experimental PTSD, male C57BL/6 mice were given 5 different foot-shocks (IFS; 1.0 mA, 1 sec duration) in 6 min. Before each shock, a tone was played to act as the PTSD-associated trigger. Control mice were also placed in the IFS chambers for 6 min but did not receive foot shocks. Behavioral testing was performed to characterize mice into non-responders (NR; IFS mice that do not demonstrate PTSD-like behavioral characteristics) and PTSD-like mice. Terminal timepoints selected were 4-weeks (4.6±0.7 months old) and 13-weeks (8.2±0.0 months old) post-IFS. Doppler echocardiography was collected at the 4-week time point. Histological and immunoblot assessments were collected at 13-weeks post-IFS to determine chronic alterations in cardiac homeostasis. Doppler measurements revealed that 4-weeks post-IFS, PTSD-like mice had decreased aortic ejection time (p<0.05) and trended toward increased isovolumetric relaxation (p=0.080) and contraction (p=0.088) time compared to controls. Interestingly at 13-weeks post-IFS, cardiomyocyte size was also elevated in PTSD-like mice compared to controls (p<0.05), suggesting the functional changes observed at 4-weeks reflect elevations in myocardial stress. A decrease in spleen weight at 4- (p<0.05) but not at 13-weeks (p=0.30) post-IFS indicate potential recruitment of immune cells acutely in PTSD-like mice. In line with spleen weights, at 4- and 13-weeks post-IFS, macrophage staining showed elevated levels in the LV of PTSD-like mice but not NR compared to controls (p<0.05 for all). In addition, matrix metalloproteinase (MMP)-9 protein levels were increased in the LV of PTSD-like mice compared to controls 13-weeks post-IFS (p<0.05). Collagen volume fraction was elevated at 13-weeks post-IFS in PTSD-like and NR mice compared to controls (p<0.05 for all). Our data indicates PTSD-induced cardiac stress is leading to macrophage recruitment and cardiac fibrosis which likely over time will lead to deterioration of myocardial function.

This work was supported by the National Institutes of Health T32GM123055; the American Heart Association Innovator Project IPA35260039; the Biomedical Laboratory Research and Development Service of the Veterans Affairs Office of Research and Development Award IK2BX003922; and South Carolina Translational Research Center UL1TR001450

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.
Hyperinsulinemia alters Notch1 and ZO-1 expression in human brain microvascular endothelial cells
Authors: Khan Sabina , Altman Robin
Published: May 2023

Alzheimer’s disease (AD) is an irreversible disease of aging that progressively impairs cognitive function. Although the pathogenesis of AD remains elusive, dysfunction of the blood-brain barrier (BBB) appears to be an important contributing factor. Of the cells that form the BBB, brain microvascular endothelial cells play an important role in maintaining the integrity of the barrier. Disruption of endothelial tight junctions may contribute to the BBB dysfunction observed in AD. Complete understanding of how this may occur remains unclear, but the intersection between AD and type 2 diabetes mellitus (T2DM) suggests possibilities. Individuals with T2DM are at greater risk of developing AD, and the hyperinsulinemia that characterizes untreated T2DM may contribute to endothelial damage and BBB dysfunction by disrupting tight junctions. The cellular mechanisms by which this dynamic occurs continue to be explored, but one signaling pathway that may be involved is the Notch signaling pathway. The objective of this study was to investigate the effects of hyperinsulinemia on Notch1 and zonula occludens-1 (ZO-1, a key tight junctional protein) expression in human brain microvascular endothelial cells. We hypothesized hyperinsulinemia disrupts tight junctions in brain endothelial cells by decreasing ZO-1 protein expression in a Notch1-dependent manner. To explore this hypothesis, human cerebral microvascular endothelial cells (hCMEC/d3 cell line) were exposed to hyperinsulinemic conditions (100 nM) for 0.5, 1, 2, and 4 hours. Following treatment, total cellular protein was isolated, quantified, and separated by SDS-PAGE. Notch1 and ZO-1 protein expression were analyzed by western blot, and expression levels normalized to GAPDH. Our results indicate hyperinsulinemia increased protein expression of Notch1 by almost 5-fold and decreased ZO-1 expression by 3-fold in a time-dependent manner compared to untreated control cells. These results demonstrate hyperinsulinemia alters both Notch1 and ZO-1 protein expression along a parallel timeline, suggesting ZO-1 expression may be dependent on Notch1 signaling; however, further studies are needed to determine whether Notch1 signaling is required for decreased ZO-1 expression during hyperinsulinemic conditions. Disruption of cerebral microvascular endothelial tight junctional integrity due to hyperinsulinemia in T2DM may negatively impact BBB functionality, lending insight into the development and progression of AD.

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.
Bioflavonoid luteolin normalizes pathways implicated in preeclampsia vascular dysfunction
Authors: Eddy Adrian , Spradley Frank , Rajakumar Augustine , Granger Joey , Rana Sarosh
Published: May 2023

Preeclampsia (PE) is a pregnancy disease characterized by maternal hypertension and vascular dysfunction. PE is believed to originate from the ischemic/hypoxic placenta that activate local HIF-1α-mediated release of anti-angiogenic sFlt-1 and release of inflammatory cytokines, such as TNF-α. Signaling from both sFlt-1 and TNF-α-induced NF-κB activation result in upregulation of the potent vasoconstrictor ET-1. Antagonism of ET-1 signaling has been shown to relieve symptoms in animal models of PE, but these treatments are contraindicated in pregnancy. Luteolin is a naturally occurring bioflavonoid that has been studied for its use as an anti-inflammatory and anti-hypertensive agent but has never been studied in PE. In this in vitro study, we tested the hypothesis that luteolin may attenuate pathways involved in vascular dysfunction by attenuating HIF-1α increase (via upstream PI3K and ERK) as well as TNF-α-induced NF-κB activation. To study HIF-1α inhibition by luteolin, placental explants from normotensive patients were treated under normoxic (20%) and hypoxic (2%) conditions with luteolin (10μM) for 72 hours, in the presence and absence of relevant inhibitors; PI3K inhibitor (LY294002, 50μM) and ERK inhibitor (U0126, 20μM). Western blots were used to measure HIF-1α protein changes. HUVEC cells were stimulated by TNF-α in the presence or absence of luteolin to determine NF-κB nuclearization by immunofluorescence.Hypoxic incubations significantly increased HIF-1α compared to normoxia (1.44 + 0.43 vs 0.55 + 0.21 arbitrary units (AU); p<0.001). Although HIF-1α expression remained unchanged regardless of treatment in normoxic samples, hypoxic samples treated with luteolin had a significant reduction of HIF-1α expression (1.44 + 0.43 vs 0.51 + 0.09 AU; p<0.01). Similarly, HIF-1α expression was reduced by PI3K inhibitor treatment in hypoxia (1.44 + 0.43 vs 0.56 + 0.18 AU; p<0.01) and ERK 1/2 inhibitor (1.44 + 0.43 vs 0.66 + 0.24 AU; p<0.05). The combination of luteolin and inhibitors did not result in an additive effect. NF-κB nuclearization increased significantly after stimulation with TNF-α (0.38 + 0.13 vs 1.07 + 0.45 AU; p<0.01), which was ameliorated by luteolin to 0.51 + 0.18 AU (p<0.05).Maternal vascular dysfunction is a final common pathway in the pathogenesis of PE, stimulated by upregulation of ET-1. Signaling by ET-1 is promoted by upregulation of HIF-1α-induced sFlt-1 and TNF-α activation of NF-κB. Luteolin, based on our data, is capable of targeting these pathways of ET-1 upregulation and has the potential to serve as a therapeutic for PE.

This work was supported by NIH 1R56HL157579-01.

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.
Age-related changes in cardiac contractility in a mouse model of Down syndrome
Authors: Vasileva Anastasiia , Vasilyev Mikhail , Tomasson Michael , DeRuisseau Lara , Bates Melissa
Published: May 2023

Over the last couple of decades, the median survival of individuals with Down syndrome (Ds) increased from 30 to 60 years. The Ts65Dn mouse model recapitulates most of the cognitive and neural phenotypes described in Ds individuals and its use continues to expand into other fields. The Ds population is at risk for cardiovascular comorbidities. We hypothesized that contractility in Ts65Dn mice will be lower compared to controls, and differences between strains will become exacerbated with age. The aim of this study was to comprehensively access the cardiac function in the Ts65Dn model across the lifespan. Ts65Dn and C57BL/6J mice (n = 3-5) were tested at 3, 6, and 12 months of age. Left ventricular function was assessed in closed-chest mice using a microtip pressure-volume catheter (PVR-1030, Millar) which was introduced via the right carotid artery and coupled to a digital converter (ADInstruments). By transiently compressing the inferior abdominal vena cava, different preloads were induced, accessing hemodynamic assessment of the left ventricle. Finally, the heart was excised, trimmed, & weighed to assess the Fulton index. The end-diastolic pressure-volume relationship (ESPVR) increased at 12 months in Ts65Dn mice compared to controls. Comparison of the maximal velocity of pressure rise (+ dP/dt) did not differ between ages and genotypes. The maximal velocity of pressure fall (− dP/dt), however, was significantly lower in 12 months Ts65Dn compared to the 6-month group. Cardiac output (Q̇) decreased with aging in Ts65Dn mice compared to controls, therefore decreasing stroke volume (SV) and stroke work (SW). The index of arterial elastance (Ea) increased with aging in Ts65Dn mice compared to the control. Together, Ts65Dn mice have decreased systolic performance accompanied by increased contractility of the heart and increased aortic elastance, and these parameters are revealed as mice age.

NIH R21HD099573

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.
Skeletal muscle transcriptomic differences underlie blunted mitochondrial adaptions following combined aerobic exercise and metformin
Authors: Elliehausen Christian , Minton Dennis , Lande Kat , Spiegelhoff Audrey , Konopka Adam
Published: May 2023

The multi-morbidity of aging limits the value of targeting any single age associated disease. Despite exercise and metformin individually improving physiological hallmarks of aging, we and others have shown that metformin inhibits adaptions to insulin sensitivity, skeletal muscle mitochondrial respiratory capacity, and cardiorespiratory fitness after aerobic exercise training (AET). The mechanisms underlying these antagonistic effects are unknown but highlight an important need to further understand the interaction between two leading health span treatments. Here, we performed an 8-week progressive AET regimen on 8-month-old male C57BL6/J mice with metformin (AET+MET) in the drinking water (~300mg/kg/day), without (AET), and sedentary control (SED) (n=3/group). Using high resolution respirometry with permeabilized muscle fibers, metformin prevented the increase in complex I (CI) linked respiration after AET. Additional analyses of site-specific hydrogen peroxide (H2O2) emissions in isolated muscle mitochondria revealed metformin increased H2O2 emissions at site IF of CI versus AET with no difference at site IQ. In this preliminary data set, we did not detect differences between AET or AET+MET in glucose tolerance, insulin sensitivity, or maximal exercise performance following exercise. To facilitate the development of hypothesis generating questions, we aimed to identify a skeletal muscle gene expression prolife that was associated with the effects of metformin on skeletal muscle mitochondrial bioenergetics. Globally the number of differentially expressed genes was blunted by the addition of metformin to aerobic exercise training. Both the upregulation (AET: 139 genes vs. AET+MET: 45) and down regulation of genes (AET: 138 genes vs. AET+MET: 108) was attenuated by the addition of metformin. We identified Nicotinamide Riboside Kinase 2 (NMRK2) as the greatest differentially expressed gene between AET and AET+MET. NMRK2 has a dual role in NAD+ biosynthesis and integrin signaling. Therefore, metformin may alter the transduction of mechanical stimuli to NAD+ homeostasis via NMRK2. While the current study is limited in sample size, we have an ongoing cohort to validate these initial findings. Collectively, our data suggest metformin inhibits skeletal muscle mitochondrial adaptations of aerobic exercise training, similar to our previous work in humans. Further, we have identified NMRK2 as a potential candidate mechanism that is associated with how metformin mediates mitochondrial adaptations to exercise.

Nathan Shock Center, Salk Institute for Biological Sciences

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.
SGLT2 Inhibition Alters the Hepatic and Circulating Metabolome of Mice Under a High-Milk-Fat Diet
Authors: Mendez Espinoza Ileana , Shepard Blythe
Published: May 2023

Recent studies have shown that the inhibition of Sodium-glucose cotransporter 2 (SGLT2), which reabsorbs glucose in the renal proximal tubule, can aid in glycemic control under conditions of type 2 diabetes (T2D). Though it acts directly on the kidney, these inhibitors also lead to great improvement in liver function. Previously, we challenged TallyHo mice, an inbred polygenetic mice model that recapitulates T2D, with a high milk fat diet (HMFD) for 12 weeks in the continued absence or presence of Empagliflozin (EMPA), an FDA-approved SGLT2 inhibitor. Male mice showed a decrease in circulating triglycerides, hyperglycemia, and hepatic lipid and cholesterol esters (Kurtz et al 2022). However, the mechanisms that underlie this improved liver phenotype is unknown. Thus, we sought to characterize the hepatic and circulating metabolic and lipidomic profiles that are altered and/or restored in the presence of EMPA using targeted and untargeted LC-MS. Control, HMFD, and HMFD + EMPA liver tissues and plasma samples were dissolved in a methanol/water 50/50 extraction buffer that contained debrisoquine (DBQ) for the positive mode standard and 4-nitrobenzoic acid for the negative mode standard. For the untargeted liver and plasma samples, a UPLC-QTOF analysis was performed. MetaboAnalyst was used to quantify metabolite changes with inclusion criteria set to a fold change of 2 and a p-value <0.05. Of note, previous studies have shown that EMPA promotes the upregulation of ketone bodies including β-Hydroxybutyrate (BHB). We noted a similar upregulation of BHB in our metabolomic analysis and confirmed that EMPA led to a ~1.5-fold increase (p < 0.022) in this circulating ketone body. In addition, of those metabolites that met the inclusion criteria, we identified 12 that were downregulated and 9 that were upregulated +EMPA in the male samples. Out of these, orotate, sarcosine, tartrate, and dihydrofolate levels were restored to those of control levels. In the female samples, 6 were downregulated and 3 were upregulated +EMPA. Indoxyl sulfate, butylcarnitine, and valerylcarnetine were all restored to control levels. Of particular interest is the finding that EMPA treatment restores levels of key metabolites that are involved in the folate and pyrimidine biosynthesis pathways. Previous studies have determined that folate deficiency can promote insulin resistance and the urea cycle, a pathway that is integrated with pyrimidine biosynthesis, is upregulated under conditions of T2D and non-alcoholic fatty liver disease. Future studies are aimed at identifying the role of EMPA's hepatic protection in these metabolic pathways.

NIDDK

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.
Rapid, activity-dependent co-release of GABA and glycine in the mouse brainstem
Authors: Gao Hong , Desmoulins Lucie , Molinas Adrien , Zsombok Andrea , Derbenev Andrei
Published: May 2023

Presympathetic neurons in the ventrolateral and ventromedial medulla (VLM/VMM) control the sympathetic tone, and GABA was identified as a primary mechanism for sympathoinhibition. Intriguingly, there is evidence that both GABA and glycine efficiently inhibit sympathetic tone; however, the mechanism of glycine release is unknown. In this study, we tested the hypothesis that GABA and glycine are co-released in the VLM/VMM. Glycine is recycled by glycine transporter 2 (GlyT2), which is a reliable marker of glycinergic neurons. Therefore, we crossed heterozygous GlyT2Cre mice with floxed channelrhodopsin 2 (ChR2-EYFP) mice to generate GlyT2 reporter mice (GlyT2ChR2/EYFP), which were used to determine the mechanism of glycine release and reveal the location of glycinergic neurons. Whole-cell patch-clamp recordings from presympathetic VLM/VMM neurons revealed that GABA mediates most spontaneous inhibitory postsynaptic currents (sIPSCs); whereas increased activity of inhibitory inputs promotes the release of glycine. Light stimulation of glycinergic fibers expressing ChR2 in GlyT2ChR2/EYFP mice evoked IPSCs (eIPSCs) with glycinergic and GABAergic components. Bath application of GABAA receptors antagonist (bicuculline, 10 μM) decreased the amplitude of eIPSCs, suggesting that approximately 70% of the eIPSCs are generated by glycine. Moreover, blockade of GlyT2 partially diminished the glycinergic component of the light-evoked IPSCs. Using GlyT2ChR2/EYFP mice, we found that glycinergic fibers are abundant throughout the rostral brainstem, and GlyT2-expressing neurons were distributed within the ventral gigantocellular nucleus and medial reticular formation. Immunofluorescence labeling of glycine receptor subunits in the VLM/VMM revealed the expression of two prevalent forms of glycine receptors formed by α1 and α3. In summary, we found that GABA and glycine are co-released in the VLM/VMM, and blockage of GlyT2 decreased the amplitude of light-evoked IPSCs, suggesting that GABA and glycine are likely located in separate vesicles. Our data provide evidence for the co-inhibition of presympathetic neurons in the VLM/VMM by GABA and glycine and suggest that GABA controls the threshold excitability, while glycine is released on demand to increase the strength of inhibition.

Supported by: NIDDK122842 and Tulane Brain Institute Marko Spark Innovation Research Fund

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 novel MYH6-E1584K tail domain variant associated with hypoplastic left heart syndrome leads to hypercontractility in vitro
Authors: Anfinson Melissa , Thareja Suma , Cavanaugh Matthew , Brown Ryan , Kim Min-Su , Liang Huan-Ling , Mitchell Michael , Fitts Robert , Tomita-Mitchell Aoy
Published: May 2023

Hypoplastic left heart syndrome (HLHS) is a complex form of congenital heart disease (CHD) characterized by hypoplasia of the left ventricle and proximal aorta, as well as stenosis or atresia of the mitral and aortic valves. Our lab previously identified that rare, predicted-damaging variants in the gene encoding for α-myosin heavy chain ( MYH6, α-MHC), a key contractile protein in the heart, are enriched in HLHS. It has been shown that pathological variants in the MHC head domain directly alter force generation, but mechanisms by which tail domain variants cause contractile defects are less clear.In double-blind analyses, patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) carrying the MYH6 E1584K tail domain variant (VAR) showed significant structural and functional deficits compared to wild-type (WT). Consistent with our previously reported findings in atrial tissue from a patient carrying MYH6 E1584K, VAR iPSC-CMs exhibited sarcomere disarray (p<0.001) and altered sarcomeric gene expression (p<0.05). VAR iPSC-CMs display a hypercontractile phenotype compared to WT, with increased amplitude (2.16μm VAR, 1.32μm WT; p<0.05) and duration (233 msec VAR, 173 msec WT; p<0.05) of contraction and elongated relaxation times (270 msec VAR, 190 msec WT; p<0.01). Hypercontractility was also observed in bulk culture, where VAR iPSC-CMs demonstrated increased amplitude (1413.4 a.u. VAR, 993.6 a.u. WT, p<0.05) and velocity of contraction (1493.7 a.u./msec VAR, 982.2 a.u./msec WT, p<0.05). Notably, this is different than our previous reports of iPSC-CMs carrying the head domain variant MYH6 R443P, which exhibited decreased contraction velocity.Our data demonstrate the mechanism of MYH6 pathogenicity is distinct between variants, emphasizing the importance of mechanistic studies to inform clinical decision-making. Furthermore, this work will direct future investigations into the genetic etiology and physiology of CHD and provide a framework upon which personalized treatment strategies can be developed. Future studies will examine the effect of myosin modulators omecamtiv mecarbil (OM) and mavacamten on MYH6 variant iPSC-CMs.

Advancing a Healthier Wisconsin Endowment

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.
Supraclavicular brown adipocytes arise from myogenic Tbx1-lineage progenitors
Authors: Ruan Hai-Bin , Huang Zan , Zhang Zengdi
Published: May 2023

Brown adipose tissue (BAT) is a thermogenic organ that promotes metabolic fitness. In humans, there are two spatiotemporally distinct phases of BAT ontogeny: the prenatally developed dorsal-anterior-located interscapular BAT (iBAT); and the postnatally emerged supraclavicular BAT (sBAT). It has been established in rodents that the dorsal-anterior-located iBAT depots predominately arise from a Pax3 +/ Myf5 + lineage in the epaxial dermomyotome that also forms the trunk muscle. However, the developmental origins of the supraclavicular BAT that adult humans have remain unknown. In this study, using lineage tracing mouse models, we found that these supraclavicular BAT depots are not derived from the Pax3 + or Myf5 + precursor cells. Knock out of PPARγ, the master regulator of adipogenesis, in Myf5 + cells caused severe atrophy of iBAT, but did not affect the supraclavicular depots. Instead, we identified that most of the supraclavicular brown adipocytes are progeny cells of the developmental Tbx1 + lineage that also gives rise to neck muscle cells. PPARγ deficiency in this lineage did not perturb iBAT development, but caused paucity in the supraclavicular regions, thus sensitizing mice to cold-induced decrease in body temperature.In summary, our results suggest that there are region-specific myogenic precursors that contribute to BAT development at different anatomical locations. Future studies to understand the pathophysiological regulation of these developmentally distinct BAT depots could inspire new approaches to measure BAT mass and to activate BAT activity in humans for the treatment of obesity, diabetes and related metabolic disorders.

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 intermittent fasting on brain tissue in male Fisher-344 rats
Authors: Sudasinghe Keshari , White Zachary , Rogers Liza , Hall Stephanie
Published: May 2023

Introduction: Intermittent fasting (IF), time-restricted food intake without any caloric restrictions, has been shown to improve cognition in animal models. Several pathways have been identified in IF-induced neuroprotection; here, we observed the effect of IF on neuroprotection targeting molecular markers of protein synthesis (mTOR), autophagy (ATG5/ LC3A), and antioxidant expression (SOD2). We hypothesized that IF would improve autophagy and antioxidant expression and reduce protein synthesis in male fisher-344 rats. Methods: Twenty-four, 10-week-old male Fisher-344 rats were randomly assigned into two groups (ad-libitum, or IF) and housed 4 per cage, on a 12:12 light: dark cycle. Ad-libitum group had 24/7 access to food and water while the IF group had 24/7 access to water and full access to food on an every-other-day basis (i.e. alternate day fasting) for ten weeks. At 20 weeks of age, animals were euthanized and brain tissues (hippocampus and cortex) were sectioned and snap-frozen in liquid nitrogen. Following homogenization, western blots were completed via Jess Protein Simple automated western blotting (Bio-Techne, Minneapolis, MN). Specific proteins of interested included, LC3 (Novas, NB100-2331), ATG5 (Novas, NB110-538185S), mTOR (Santa Cruz, sc-517464) and SOD2 (Abcam, ab68155). Compass software generated area under the curve values were used to determine protein expression. Group differences were analyzed with a two-tailed T-test per Prism Graph Pad software. Results: In cortex tissue, a significantly lower mTOR expression was observed in the IF group compared to the AL group (p<.001), however, mTOR expression was not significantly different in the hippocampus. Antioxidant enzyme, SOD2, was significantly higher in IF cortex tissues (p<.05) compared to AL controls. In contrast, SOD2 was significantly lower in IF hippocampal tissues (p<.01) compared to controls. No significant effect was documented in the LC3 and ATG5 autophagy markers. Conclusion: In the present study, IF significantly impaired protein synthesis (via mTOR expression) in the cortex only. Previous work has also concluded the downregulation of mTOR with both calorie restriction and fasting. However, the fact that no decline was present in the hippocampus is intriguing and will be the focus of follow-up investigations. As expected, IF increased antioxidant enzyme (SOD2) expression in the cortex and interestedly, IF decreased antioxidant expression in the hippocampus. While the hippocampus is noted for its high antioxidant content, this decreased expression induced with IF warrants more research.

P20GM113109

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.
Decreased arterial elastance following a novel RSK3/mAKAPß gene therapy is associated with improved bradykinin-dependent pial artery dilation in aortic-banded, Western diet-fed female Ossabaw swine
Authors: Tharp Darla , Amin Amira , Thorne Pamela , Mueller Christina , Li Jinliang , Olver T. Dylan , Kapiloff Michael , Emter Craig
Published: May 2023

Cardiogenic dementia is associated with increased risk of mortality in heart failure. Cognitive decline is more prevalent in females, of which cardiac function is a strong predictor. We have demonstrated cerebrovascular insufficiency in female Ossabaw swine with cardiometabolic heart failure (CM-HF). The purpose of this study was to examine the effect of a novel gene therapy that displaces p90 ribosomal S6 kinase 3 (RSK3) from perinuclear muscle A-kinase anchoring protein β (mAKAPβ) signalosomes using a systemically delivered adeno-associated virus (AAV) to express a RSK3/mAKAPβ anchoring disruptor peptide (RBD) controlled by a cardiomyocyte-specific promoter (AAV9.RBD). We hypothesized that disruption of RSK3/mAKAPβ by AAV9.RBD would improve cardiac and cerebrovascular function in parallel, diminishing cardiogenic pathology. Female Ossabaw pigs were assigned to HF control (HF; n=4) and HF AAV9.RBD-treated (HF+RBD; n=5) groups. Animals were fed a Western diet (3 mo. old) and aortic banded (6 mo. old) prior to euthanasia (12 mo. old). Pressure-volume hemodynamics were used to measure arterial elastance (Ea), the ratio of end systolic pressure:stroke volume (SV). Pressure myography was used to assess in vitro cerebral vascular function (2a pial arterioles; inner diameter 267 ± 19 μm HF & 248 ± 9 μm HF+RBD). Group comparisons were made using student’s t-test and repeated measures ANOVA with significance reported at the P≤0.05 level. Ea was decreased in HF+RBD animals compared to HF (mmHg/mL; 2.3 ± 0.2 HF & 1.7 ± 0.2 HF+RBD) and driven by increased SV (mL; 40 ± 2 HF & 50 ± 2 HF+RBD). Decreased arterial load was associated with increased endothelial-dependent dilation to bradykinin (BK) in the HF+RBD group compared to HF (Group x Dose interaction) in isolated pial arteries. BK-induced dilation was decreased in the presence of L-NAME (nitric oxide synthase inhibitor) in HF, but not HF+RBD animals (Group x Dose interaction). In contrast, BK-induced dilation was decreased in the presence of indomethacin (prostaglandin synthesis inhibitor) only in the HF+RBD group (Group x Dose interaction). In conclusion, decreased cardiac arterial load (Ea) was coupled with improved endothelial-dependent cerebral vascular function that was mechanistically characterized by altered nitric oxide/prostaglandin balance in female Ossabaw swine with CM-HF. These findings suggest AAV9.RBD-mediated disruption of RSK3/mAKAPβ signalosomes in cardiomyocytes has a positive cardiogenic effect on cerebrovascular function.

Department of Defense (DOD) W81XWH-18-1-0179

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.
Macrophage to Myofibroblast Transition in Diastolic Dysfunction Development
Authors: Mora Christoph , Liu Yunmeng , Benson Lance , Deck Katherine , Guo Yunping , Mu Shengyu
Published: May 2023

Heart failure is the top killer in the U.S and worldwide, often caused by hypertension, aging, or cardiac toxicity. Diastolic Dysfunction (DD), the often the first step of heart failure, occurs when the chambers of the heart are not able to expand during the filling phase. In addition, hypertension is the leading cause of DD. Though this is known, the progression of hypertension into DD remains poorly understood. Of note is the ability of M2 macrophages to differentiate into a myofibroblast-like phenotype, known as macrophage to myofibroblast transition (MMT). This transition has been observed in interstitial fibrosis in chronic renal allograft injury. The researchers noted that MMT account for 50% of the myofibroblast population. To our knowledge, the extent of fibrotic remodeling due to MMT in the heart has not been ardently investigated. Therefore, we aim to answer whether MMT plays a causal role in the progression of hypertension to DD and the pathogenesis of heart failure.Utilizing the uninephrectomized, DOCA-salt model, mice were subjected to this treatment and hypertension was induced. As a negative control, another group of mice received a uninephrectomy and the same salt in their drinking water, however, DOCA was not administered (Sham group). MMT prevalence, identified as CD68+ α-SMA+ expressions, was observed inside the hearts of DOCA and Sham mice. The hearts were isolated and homogenized via gentleMACS tissue Dissociator, the monocytes were purified via Percoll isolation, and flow cytometry was used to identify the population of CD68+ α-SMA+ expressing cells.As soon as 10 days post DOCA implantation, the DOCA group exhibited worsening diastolic function as it took longer for the pressure to change in the hearts of DOCA mice than in Sham (-dp/dt). These data were gathered via left ventricular (LV) catheter insertion. At a later time, utilizing a different cohort of mice preliminary data show an increase of monocyte (CD68+) and fibroblast (α-SMA+) double-positive expressing cells in the hearts of hypertensive mice.MMT has been observed in the hearts of hypertensive mice. This transition may exacerbate Diastolic Dysfunction. More research must be performed to investigate the wide variety of functions of macrophages (i.e., M1 to M2 ratio and transition, macrophage residence or infiltration, and macrophage stimulation of myofibroblasts), as well as the specific interactions between macrophages and cardiomyocytes, or other immune cells, that might mediate this transition. These data provide a preliminary, but interesting glimpse, into the role of macrophages in exacerbating Diastolic Dysfunction and mediating Heart Failure.

NIH RO1-HL146713 and AHA 15BGIA25730047 (Shengyu Mu)

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.
Dietary-induced changes in the hindgut microbiome and metabolism of a marine herbivorous fish
Authors: Herrera Michelle , Heras Joseph , Catabay Caitlyn , Connor Kwasi , German Donovan
Published: May 2023

To advance our understanding of the factors that influence gut physiology and function, we are examining how the gut microbiome changes under varying environments and diets. We integrated the gut transcriptome and microbiome to understand how dietary changes impact Cebidichthys violaceus, an herbivorous fish native to the California coast that is known to have an active microbial community in its hindgut. Wild-caught fish from San Simeon, California were fed either herbivore, omnivore, or carnivore diets in the laboratory for six months. We expected the microbiome, as well as the hindgut and liver transcriptome, to change with dietary perturbations, with wild herbivorous fish being most similar to lab-herbivore fish. Wild fish microbial communities were indeed different from laboratory-fed fish (P<0.001, Bray-Curtis distances), but the laboratory-omnivore diet fish were the most similar to wild fish, sharing a high abundance of taxa in the Bacteroidota and Bacillota (Families Ruminococcaceae and Rikenellaceae), whereas the laboratory-herbivore and laboratory-carnivore fish shared a high abundance of taxa from the Pseudomonodota (Families Burkholderiaceae and Oxalobacteraceae). The microbial diversity patterns in multivariate space among the fish on the different diets directly resembled that of host distal intestine gene expression in multivariate space (i.e., wild and lab-omnivore diet fish were the most similar). The hindgut had 519 differentially expressed genes, and wild fish highly express genes involved in ion transport and lipid and glucose metabolism. The liver, on the other hand, presented 4650 differentially expressed genes, and wild fish highly express genes for fatty acid synthesis and proteolysis. These data show how the gut microbiome and transcriptome varies as a function of different diets in an herbivorous marine fish. Our project provides insight into what it takes for fish to deal with shifting resources, which is important for marine resource management and new potential aquaculture species development.

NSF Grant IOS-1355224 and William Townsend Porter Pre-doctoral Fellowship from the American Physiological Society.

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.