bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibiting renal Traf2 and NCK interacting kinase (TNIK) decreases NKKC2 phosphorylation and blunts salt-sensitive hypertension
Authors: Granados Jessica , Caceres Paulo , Ortiz Pablo
Published: May 2024

The Na/K/2Cl cotransporter NKCC2 mediates the NaCl reabsorption in the thick ascending limb (TAL). Phosphorylation at Thr-96,101 and traffcking to the surface are increased in the Dahl salt-sensitive (DSS) rat together with SPAK phosphorylation, an upstream kinase for Thr-96,101 in NKCC2. We previously found that deletion of SPAK in the DSS, lowers NKCC2 phosphorylation and blunts salt-sensitive hypertension, but doesn’t completely abolish NKCC2 phosphorylation. Therefore, other kinases may be involved in NKCC2 phosphorylation. We recently found the Traf2 and NCK interacting kinase (TNIK) phosphorylates NKCC2 and is overexpressed in the TALs of DSS rats. We hypothesized that TNIK contributes to salt-sensitive hypertension by increasing NKCC2 hyperphosphorylation in DSS rats. We approached TNIK inhibition in two ways, using a pharmacological inhibitor and its genomic deletion with CRISPR/Cas9. First, we studied the small molecule TNIK inhibitor NCB-0846, and whether it could decrease baseline NKCC2 phosphorylation in TALs from DSS rats. Treating TALs from DSS with NCB-0846 (0.1 μM) for 25 min decreased NKCC2 Thr-96,101 phosphorylation by 21 ± 2% compared to the vehicle group (p<0.001, n=3). To study in vivo effcacy of NCB-0846, we directly infused NCB (or vehicle) into the left kidney via implanted renal medullary catheters connected to minipumps in DSS rats fed 4% NaCl diet. We found that NCB-0846 decreased SBP by 30 mmHg within four days compared to the vehicle-infused group, and BP remained lower for 7 days (n=5, p<0.03 vs control). To decrease TNIK gene expression we used Cas9 mediated gene editing. For this, we designed gRNA sequences targeting TNIK and tested their effcacy in C6 cells. One of the gRNAs designed decreased TNIK expression by 48 ± 22% compared to control (p=0.101, n=3). To decrease TNIK in TALs in vivo, we injected the renal medulla of the left kidney with AAV-gRNA-TNIK together with AAV-NKCC2 promoter-Cas9. After 2 weeks, we isolated medullary TALs and measured TNIK expression and NKCC2 phosphorylation. TNIK expression was decreased by 50 ± 5.5% (n=4, p<0.0001), and pThr-96,101-NKCC2 was decreased by 38.1 ± 10.6% in transduced TALs compared to kidneys transduced with AV-control (n=4, p<0.05). The expression of related kinases SPAK and OSR1 was not modified (SPAK 79.2 ± 18.3%, OSR1 81.3 ± 13.3%, n=4, ns). We conclude that NKCC2 phosphorylation in the DSS rat is in part caused by TNIK and that this new kinase could be a target for salt sensitive hypertension. Future experiments will test the effect of gene editing TNIK in salt sensitive hypertension.

23POST1019882 AHA15GRNT25710369.

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 L-PGDS in Light Alcohol Consumption-Induced Cerebral Angiogenesis in Male Mice
Authors: Sun Hong , Li Jiyu , Li Chun , Subedi Pushpa , Subedi Utsab
Published: May 2024

Ischemic stroke is one of the leading causes of death and permanent disability. Promoting cerebral angiogenesis before or after ischemic stroke reduces ischemic brain damage and improves functional recovery. Alcohol is one of the most commonly consumed chemical substances. Light alcohol consumption (LAC) reduces the incidence and improves the prognosis of ischemic stroke. Recently, we found that LAC promotes cerebral angiogenesis under basal conditions and following ischemic stroke. In addition, LAC upregulates lipocalin-type prostaglandin D2 synthase (L-PGDS), the principal PGH2 isomerase mainly expressed in the central nervous and cardiovascular systems. Therefore, we determined the role of L-PGDS in LAC-induced cerebral angiogenesis. In in vitro studies, low-concentration ethanol significantly upregulated L-PGDS and increased angiogenic capability (tube formation, migration, and proliferation) in cultured C57BL/6J mouse brain microvascular endothelial cells (MBMVECs). AT-56, a selective L-PGDS inhibitor, abolished the increased angiogenic capability. In in vivo studies, 8-week gavage feeding with low-dose ethanol significantly upregulated L-PGDS and increased vessel density and vessel branches in the cerebral cortex and subcortical area under physiological conditions and following ischemic stroke in C57BL/6J male mice. AT-56 and endothelial cell-specific L-PGDS conditional knockout did not affect vessel density and vessel branches of the cerebral cortex and subcortical area in water-fed control mice but alleviated the proangiogenic effect in low-dose ethanol-fed mice under basal conditions and following ischemic stroke. Therefore, our findings suggest that L-PGDS may be crucial in LAC-induced cerebral angiogenesis.

NIH 2R01AA023610.

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.
Molecular Characterization of the Ca2+ Release Activated Ca2+ Current in Native Brain Capillary Endothelial Cells
Authors: Sanchez Alfredo , Lavanderos Boris , Trebak Mohamed , Earley Scott
Published: May 2024

Brain capillary endothelial cells (cECs) form the blood-brain barrier orchestrate on-demand increases in blood flow to serve the brain’s unique metabolic needs. An in vivo imaging study showed that brain capillaries generate an array of dynamic Ca2+ signals primarily dependent on the release of Ca2+ from the endoplasmic reticulum (ER). Little is known about the physiological functions and origins of these signals. Store-operated Ca2+ entry (SOCE) is a fundamental Ca2+ influx mechanism activated by emptying ER Ca2+ stores. Following store depletion, interactions between the ER Ca2+-sensor stromal interaction molecule 1 (STIM1) and Orai1, 2, and/or 3 ion channel subunits on the plasma membrane initiate the Ca2+-release activated Ca2+ current (ICRAC) and SOCE. ICRAC and SOCE in brain cECs are uncharacterized. Therefore, we investigated STIM and Orai expression and the biophysical properties of ICRAC in primary brain cECs. Using droplet digital PCR, we found that native brain cECs express Stim1, Stim2, Orai1, and Orai3 but not Orai2. Conventional whole-cell patch-clamp electrophysiology was used to record ICRAC from freshly isolated brain cECs. Currents were induced by including the slow Ca2+ buffer EGTA in the intracellular solution. In contrast to the strong inward rectification of canonical ICRAC, currents recorded from native brain cECs were dually rectifying. The inward and outward components were blocked by La3+, enhanced with 2-APB, and inhibited with the selective Orai1 channel antagonist Synta66. Both ICRAC components were increased when a divalent-free solution (Ca2+ replaced with Cs+) was used. To gain insight into the molecular composition of CRAC channels in brain cECs, we generated EC-specific knockout (ecKO) Stim1, Orai1, and Orai3 mice. ICRAC was absent in cECs obtained from Stim1-ecKO mice. In contrast, ICRAC recorded from Orai1-ecKO mice displayed strong inward rectification with no outward current and was insensitive to Synta66. ICRAC recorded from Orai3-ecKO mice were also inwardly rectifying but were highly sensitive to Synta66. ICRAC was undetectable in cells from Orai1-Orai3-ecKO mice. These data suggest that native ICRAC in brain cECs requires the expression of Stim1, Orai1, and Orai3. The outward component of native ICRAC in these cells requires the expression of Orai1 and Orai3, but the expression of either Orai1 or Orai3 alone is suffcient to maintain the inward component.

R35HL155008, R33NS115132, and P20GM130459 (to SE).

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.
Early life stress-induced aortic endothelial dysfunction is mediated by endothelium-specific HDAC9
Authors: Kelly Gillian , Kellum Cailin , Ho Dao , Colson Jackson , Walker Savannah , Weintraub Neal L. , Kovacic Jason
Published: May 2024

Early life stress (ELS) is highly prevalent worldwide and has been associated with an elevated risk of developing cardiovascular disease (CVD) in adulthood. We previously showed that maternal separation with early weaning (MSEW), an established ELS model of neglect in mice, leads to blunted endothelium-dependent relaxation as well as increased histone deacetylase (HDAC) activity. We hypothesized that specific HDAC isoform(s) mediate the ELS-induced endothelial dysfunction. In a screen of all HDAC isoforms, HDAC1, 6, and 9 gene expression were significantly elevated in aortas from male adult MSEW and normally reared (NR) C57BL6/J mice (N=3/group, p<0.05). Western blot analysis found that only HDAC9 protein abundance was significantly elevated in aortas from male MSEW as compared to NR mice (N=6/group, p=0.01). Detection of HDAC9 via immunohistochemistry was greater in the aortic endothelium of MSEW mice compared to NR (N=5/group). Subsequently, vascular reactivity was measured with wire myography on tamoxifen inducible endothelium-specific HDAC9KO (Cdh5CreERT2-HDAC9) MSEW and NR adult mice to determine the role of HDAC9 in MSEW-induced endothelial dysfunction. Male mice exposed to MSEW displayed blunted aortic endothelial-dependent relaxation to acetylcholine compared to NR, whereas endothelial-specific knockout of HDAC9 (HDAC9KO) restored endothelial-dependent relaxation (NR: Emax=89.27%±4.15, N=5; MSEW: Emax=48.12%±22.12, N=2; NR HDAC9 KO: Emax=68.98%±4.73, N=5, MSEW HDAC9KO: Emax=70.50%±1.85, N=3). Females demonstrated no difference between MSEW and NR endothelial dependent relaxation, however, HDAC9KO MSEW displayed blunted relaxation compared to all other groups (NR: Emax= 77.14%±4.86, N=5; MSEW: Emax=84.30%±8.71, N=2; NR HDAC9 KO: Emax=70.80%±5.87, N=5, MSEW HDAC9KO: Emax=69.76%±9.10, N=4). Endothelial-independent relaxation to sodium nitroprusside (SNP) was similar in all groups in both sexes (Male: NR: Emax=96.92%±1.26, N=5; MSEW: Emax=100%±0, N=2; NR HDAC9 KO: Emax=96.52%±3.484, N=5; MSEW HDAC9KO: Emax=100%±0, N=3; Female: NR: Emax=98.58%±0.42, N=5; MSEW: Emax=100%±0, N=2; NR HDAC9 KO: Emax=98.48%±1.08, N=5; MSEW HDAC9KO: Emax=99.15%±0.85, N=4). These findings suggest that HDAC9 mediates ELS-induced endothelial dysfunction in males, however, HDAC9 may play a protective role in females. Future studies aim to further investigate sex-dependent differences in the specific role of HDAC9 in ELS related CVD.

NIH P01 HL158500, F31HL165863-01, T32GM135028.

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.
Effects of Heavy Alcohol Use on Nighttime Blood Pressure, Sleep Quality, and Urine Catecholamines
Authors: Haun Tabitha , Chang Keng-Yu , Phan Kelly , Gil Alfredo , Pham Jonathan , Truong Preston , Liu Zhaoli
Published: May 2024

Heavy alcohol drinking habits contribute to the development of hypertension; this is partly due to overactivation of the sympathetic nervous system and the increased release of catecholamines. Recently, heavy alcohol drinking has been linked to poor sleep quality, but the underlying mechanisms remain unclear. We postulated that long-term frequent heavy alcohol use could increase catecholamine levels and cause blood pressure dysregulation at night, altogether influencing sleep quality. Therefore, the purpose of this study is to test the following hypothesis that compared to non-heavy drinkers, heavy drinkers have a higher level of nighttime catecholamines, a higher levels of nighttime blood pressure, and poorer sleep quality. A total of 44 men and postmenopausal women non-smokers, free of major clinical diseases, were included in this study. Heavy drinkers were defined as those having a dried blood spot phosphatidylethanol (PEth, a biomarker to detect heavy alcohol drinking) level ≥ 20 ng/mL. Participants wore an ambulatory blood pressure monitor for 24 hours and catecholamines were measured from urine samples collected in the same 24-hour period. Sleep quality was measured by using the Pittsburgh Sleep Quality Index (PSQI) questionnaire. There were 21 heavy drinkers (mean±SEM for age: 58±1 and BMI: 26.2±0.7 kg/m2) and 23 non-heavy drinkers (age: 58±1 and BMI: 27.5±0.8 kg/m2; P≥0.3 vs. heavy drinkers). Compared to non-heavy drinkers, heavy drinkers had a higher nighttime systolic blood pressure (SBP), higher diastolic blood pressure (DBP), and a lower SBP dipping ratio (heavy drinkers vs. non-heavy drinkers for nighttime SBP: 118±3 vs. 109±2 mmHg, P=0.02; nighttime DBP: 70±2 vs. 65±2 mmHg, P=0.04; and SBP dipping ratio: 10.6±1.4% vs. 14.5±1.6%, P=0.04). No differences were found in nighttime urine catecholamines between heavy drinkers and non-heavy drinkers (P≥0.3). A total of 13 participants had a PSQI score higher than 5, indicating poor sleep quality; however, PSQI scores were similar between heavy and non-heavy drinkers (P=0.8). These findings indicate that heavy drinkers had a higher nighttime blood pressure and are consistent with the observation that heavy drinking is associated with an increased risk of hypertension in mid-life adults. Interestingly, the increase in nighttime blood pressure does not appear to be related to catecholamines and sleep quality in apparently healthy mid-life adults does not appear to be influenced by heavy drinking habits.

This work was supported by NIAAA AA028537 to CLH and AHA 1266805160 to TH.

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.
Functional sympatholysis of Neuropeptide Y-mediated vasoconstriction in humans
Authors: Wakeham Denis , MacNamara James , Hissen Sarah , Levine Benjamin , Hearon Christopher
Published: May 2024

Metabolic inhibition of sympathetic vasoconstriction (functional sympatholysis) is essential for adequate perfusion of skeletal muscle during exercise. Functional sympatholysis occurs primarily through post-junctional attenuation of norepinephrine and α-adrenergic-mediated vasoconstriction, though other non-adrenergic neurotransmitters are co-released with norepinephrine. Neuropeptide Y (NPY) is a neurotransmitter that elicits robust vasoconstriction and is co-released with norepinephrine during exercise in an intensity dependent manner. Evidence from animal models indicate that NPY is sensitive to metabolic inhibition, however metabolic inhibition of NPY-mediated vasoconstriction has not been tested in humans. We tested the hypothesis that NPY-mediated vasoconstriction would be attenuated during handgrip exercise in humans. In 10 healthy adults (7M:3F, age: 30±7 years, BMI: 24±5) we measured forearm blood flow (Doppler ultrasound), blood pressure (brachial artery catheter), and calculated changes in forearm vascular conductance (FVC) to local intra-arterial infusions of phenylephrine (PE; α1-agonist) or NPY (Y1R-agonist) during 1) intra-arterial infusion of sodium nitroprusside (SNP; nitric oxide donor) to serve as a non-metabolic vasodilatory control or 2) dynamic rhythmic handgrip exercise (EX; 15% maximal voluntary contraction). The vasoconstrictor responses to PE and NPY were quantified as a percent ΔFVC during each condition (SNP vs. EX) and were compared by two-way RM-ANOVA. The magnitude of sympatholysis (ΔFVC during exercise expressed as a percent of control) for PE and NPY was compared by a paired t-test. As expected, the vasoconstrictor response to PE was attenuated during handgrip exercise compared to SNP (ΔFVC: SNP: -50±24 vs. EX: -17±9 %; p=0.002). Similarly, NPY-mediated vasoconstriction was blunted during handgrip exercise compared to SNP (ΔFVC: SNP: -32±22 vs. EX: -11±7 %; p=0.029). There was no difference in the magnitude of sympatholysis between PE and NPY (PE: 68±18, NPY: 52±34 %; p=0.28). These data indicate that NPY-mediated vasoconstriction is sensitive to metabolic inhibition in humans, and the magnitude of sympatholysis is not different from α1-adrenergic vasoconstriction.

Funding: NIH R00HL153777.

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.
Human Adenovirus Serotype 5 (HAdV-5) Infection of Kidney Fibroblast Cells Disrupts Cysteine, Purine, and Unsaturated Fatty Acid Metabolism in a Dose- and Time-Dependent Manner
Authors: Sanchez Bailey-J C. , Grasis Juris A. , Ortiz Rudy M.
Published: May 2024

Kidneys partake in numerous metabolic responsibilities, notably clearance of insulin from circulation, gluconeogenesis, and reabsorption of amino acids. Viral-induced kidney damage, either acutely or chronically, can occur from direct impact on kidney cells and from systemic and local immunological responses to counter the viral infection. Thus, it is likely that metabolic dysfunction is a consequence of infections. The detriments of viral infection on kidney cell metabolism remains an unelucidated area. We used human adenovirus serotype 5 (HAdV-5) to infect human embryonic kidney cells (HEK293T) in a dose- and time-dependent manner. We hypothesized that optimal metabolic dysfunction results after 24 hours of infection and at the highest viral dose. Cells were exposed to three viral dosages (0.5,1.0,2.0 multiplicity of infection [MOI]) and metabolism assessed by untargeted metabolomics at four time points (6-,12-,24-, 36-hours post infection [HPI]). A total of 182 metabolites were identified in our study. As early as 6 hrs, the metabolic profile of infected cells exhibited downregulation of cystathionine, cysteine, homocysteine, hypoxanthine, linoleic acid, and oleic acid with upregulation of 5'-Deoxy-5'-methylthioadenosine (MTA) and adenine. Peak dysregulation for carbohydrate, cysteine, organic acid, purine, and unsaturated fatty acid-related metabolites was observed at 12 hrs. At 24 hours, tapering of the overall metabolite response was observed with further dysregulation seen at 36 hours. Across all three dosages, responses to infection were similar. Perturbation of the cysteine, purine, and unsaturated fatty acid metabolic pathways has not previously been considered with HAdV-5 infection and a commonly used cell line exhibiting properties of kidney, and historically neuronal components. Although not investigated, uncovering adenoviral impact on enzymatic activity in these pathways could elucidate more understanding of renal impairment. These data altogether suggest that substantial DNA viral infection disrupts cellular metabolic pathways that may impair renal functions but need to be evaluated in an in vivo model.

USDA HSI Educational Grant 2021-03397. Bailey-J Sanchez was supported by USDA HSI Educational Grant 2021-03397.

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.
Captopril treatment does not affect inflammatory and autophagy signaling in skeletal muscle of Zucker diabetic fatty rat
Authors: Lee Ji Heun , Reed Carter H , Arriaga Claudia , Carrillo Claudia , Rowling Matthew J. , Valentine Rudy J. , Selsby Joshua T.
Published: May 2024

Skeletal muscle plays a key role in the development of Type 2 Diabetes Mellitus (T2DM) as its large capacity to sequester glucose and contribute to the regulation of energy metabolism is compromised. Previous studies indicate that in skeletal muscle, T2DM drives inflammatory signaling and impairs autophagy. Captopril (Cap), an angiotensin-converting enzyme (ACE) inhibitor, is a common treatment for hypertension in T2DM patients; however, the impact of Cap on inflammatory signaling and autophagy in insulin resistant skeletal muscle remains ill-defined. We hypothesized that inflammatory signaling would be elevated and degradation of autophagosomes would be impaired in obese Zucker diabetic fatty (ZDF) rats, which are hyperphagic as they lack the leptin receptor. We further hypothesized that daily Cap treatment would attenuate these disturbances. At 14 weeks of age, the absolute weights of gastrocnemius (p<0.0001), tibialis anterior (p<0.0001), soleus (p=0.002), and extensor digitorum longus (EDL, p<0.0001) muscles were decreased by 23-34% in ZDF compared to lean rats but were not altered by 8 weeks of Cap treatment (0.5g/kg/day). Immunoblotting revealed that in soleus and EDL, inflammatory signaling proteins TLR4, phosphorylated (p)IKK α/β (ser176/180), pNFkB p65 (Ser 536), IkBα, TNFα, and AP1 were similar amongst all groups, whereas MyD88 was elevated in ZDF soleus (p=0.03) but was not corrected by Cap. The autophagy protein, LC3A/B I was increased 32% (p=0.01) in EDL from ZDF rats compared to Con; however, PI3K III, pBeclin1 (ser 93), pULK1 (ser555), LAMP2, LC3A/B II, and the LC3A/B II/I ratio were similar amongst groups in the soleus and EDL and were not altered by Cap. Compared to control, P62, a key autophagy adapter protein, was similar in soleus and decreased 27% in EDL (p<0.01) from ZDF rats and was not altered by Cap. Counter to our expectations, although muscle mass was lower in ZDF groups, obesity and insulin resistance in young ZDF rats were not suffcient to drive inflammatory signaling or disrupt autophagy in glycolytic or oxidative skeletal muscle, and these processes appeared to be resistant to Cap treatment in ZDF rats.

USDA-NIFA awarded to MJR (2021-67017-33937).

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.
Compromised Plasma Membrane Repair Contributes to Neuronal Cell Death and Neurodegeneration During Alzheimer’s Disease
Authors: Bulgart Hannah , Lopez Perez Miguel , Banford Kassidy , Tucker Alexis , Bonser Sidney , Wold Loren , Weisleder Noah
Published: May 2024

Alzheimer’s Disease (AD) is the most common neurodegenerative disease and involves accumulation of intracellular amyloid beta (Aβ) in the early stages of the disease. Aβ has been shown to compromise neuronal cell membrane integrity by increasing membrane permeability, elevating intracellular calcium concentrations, and Aβ directly penetrating and damaging the neuronal cell membrane. Damage to the cell membrane requires a rapid and effcient repair mechanism to restore the barrier function of the membrane and avoid cell death. Here, we aimed to study the effects of intracellular Aβ on neuronal cell membrane repair. To determine if membrane permeability is compromised in vivo, we stained 6-month FVB and APP/PS1 brain sections with anti-IgG. We observed a significant increase in IgG positivity in the APP/PS1 tissue as compared to the FVB brains. We repeated this with post-mortem frontal lobe tissue and observed a significant increase in IgG-positivity in the AD samples as compared to the non-AD. To assess the effect of neuronal membrane repair in AD patients, we treated mouse neuroblastomas (N2A) and primary mouse hippocampal neurons with non-AD and AD patient cerebrospinal fluid (CSF) at a 1:100 dilution or 1μM recombinant human Aβ42. We conducted laser damage assays and observed a membrane repair defect in these neuronal cell models. We explored the biochemical components of the CSF that may be contributing to this induced repair defect, we measured the Aβ42 by ELISA and stratified Aβ42 concentration by the corresponding CSF AUC. We observed a positive correlation between Aβ42 concentrations and reduced membrane repair capacity. We sought to determine the mechanism of the reduced membrane repair capacity by assessing the expression levels of membrane repair proteins and observed decreased dysferlin in cell models, 6-month APP/PS1 mouse models, and human postmortem tissues with high levels of Aβ. Overexpression of dysferlin in the AD cell models can increase dysferlin expression and restore membrane repair capacity in neurons treated with AD CSF samples or recombinant Aβ42. We have determined this reduction in repair capacity is due to reduced dysferlin expression, and future studies will determine the mechanism leading to dysferlin downregulation.

This work was supported by The Ohio State University College of Medicine Dean’s Discovery Grants Program.

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.
Myotube growth is associated with cancer-like metabolic reprogramming and is limited by phosphoglycerate dehydrogenase and pyruvate kinase M2
Authors: Stadhouders Lian , Smith Jonathon , Gabriel Brendan , Verbrugge Sander , Hammersen Tim , Kolijn Detmar , Vogel Ilse
Published: May 2024

Skeletal myofiber size and oxidative metabolism are inversely related. Glycolytic fast type myofibers are larger than high high oxidative, slow type myofibers. Paradoxically, despite their lower capacity for protein synthesis (lower myonucluar density and RNA content), glycolytic, low oxidative are more responsive to an anabolic stimulus. We hypothesized that glycolytic myofibers may make use of a Warburg-like metabolic reprogramming in response to an anabolic stimulus. The Warburg effect links growth and glycolysis in tumor cells to generate glycolytic intermediates for synthesis of nucleotides and amino. The aim of this study was to test whether a similar ‘glycolysis-for-anabolism’ metabolic reprogramming also occurs in post-mitotic, hypertrophying myofibers. For this purpose, we first induced C2C12 myotube hypertrophy with IGF-1. We then added 14C glucose to the differentiation medium and measured radioactivity in isolated protein and RNA to establish whether 14C had entered anabolism. We found that especially protein became radioactive, suggesting a glucose → glycolytic intermediates → non-essential amino acid(s) → protein series of reactions, the rate of which was increased by IGF-1. Next, to investigate the importance of glycolytic flux and non-essential amino acid synthesis for myotube hypertrophy, we exposed C2C12 and primary mouse myotubes to the glycolysis inhibitor 2-Deoxy-D-glucose (2DG). We found that inhibiting glycolysis lowered C2C12 and primary myotube size. Similarly, siRNA silencing of PHGDH and PKM2, key enzymes of the Warburg-like metabolic reprogramming, reduced C2C12 and primary myotube growth; whereas retroviral PHGDH overexpression increased C2C12 myotube size. Together these results suggest that glycolysis is important for hypertrophying myotubes, which reprogramme their metabolism to facilitate anabolism, similar to cancer cells.

Brendan M. Gabriel was supported by fellowships from the Novo Nordisk Foundation (NNF19OC0055072) & the Wenner-Gren Foundation, an Albert Renold Travel Fellowship from the European Foundation for the Study of Diabetes, and an Eric Reid Fund for Methodology from the Biochemical Society. Abdalla D. Mohamed was funded initially by Sarcoma UK (grant number SUK09.2015), then supported by funding from Postdoctoral Fellowship Program (Helmholtz Zentrum München, Germany), and currently by Cancer Research UK.

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.
Epidermal Growth Factor Reduces Collecting Duct Water Permeability by Inhibiting Aquaporin-2 Phosphorylation at Ser269
Authors: Chou C-L , Limbutara K. , Kao A.R. , Clark J.Z. , Nein E.H. , Viswanathan R. , Knepper M.A.
Published: May 2024

Prior studies showed that epidermal growth factor (EGF) inhibits vasopressin-stimulated osmotic water permeability in the renal collecting duct. Here, we investigated the underlying mechanism of this response. Using the Burg isolated perfused tubule technique in microdissected rat inner medullary collecting ducts (IMCDs), we found that addition of 0.1 μM EGF to the peritubular bath significantly decreased 0.1 nM 1‐deamino‐8‐D‐arginine vasopressin (dDAVP)-stimulated water permeability, confirming prior observations. The inhibitory effect of EGF on dDAVP-stimulated water permeability was associated with a reduction in intracellular cyclic AMP levels and protein kinase A (PKA) activity. Using phospho-specific antibodies and immunoblotting in IMCD suspensions, we showed that EGF significantly reduces dDAVP-stimulated aquaporin-2 (AQP2) phosphorylation at S264 and S269. This effect was absent when membrane-permeable 8-cpt-cAMP (0.1 mM) was used to stimulate AQP2 phosphorylation, suggesting that EGF’s effect was on a pre-cAMP step. Immunofluorescence labeling of microdissected IMCDs showed that EGF significantly reduced apical AQP2 abundance in the presence of dDAVP. To address what protein kinase might be responsible for S269 phosphorylation, we used Bayesian analysis to integrate multiple -omic datasets. Thirteen top ranked protein kinases were subsequently tested by in vitro phosphorylation experiments for their ability to phosphorylate AQP2 peptides using a mass spectrometry readout. The results show that the PKA catalytic-α subunit increased phosphorylation at S256, S264, and S269. None of the other kinases phosphorylated S269. Additionally, PKA inhibitors, H-89 and PKI, strongly inhibited dDAVP-stimulated AQP2 phosphorylation at S269. These results indicate that EGF decreases the water permeability of the collecting duct by inhibiting cAMP production, thereby inhibiting PKA and decreasing AQP2 phosphorylation at S269, a site previously shown to regulate AQP2 endocytosis.

This work was funded by NHLBI project ZIA-HL001285 and ZIA-HL006129, M.A.K.

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.
Untangling the Impacts of Ethanol on Myoblast Proliferation
Authors: Levitt Danielle , Helmberger Rylea , DiLeo Matthew
Published: May 2024

Alcohol-mediated myopathy frequently accompanies chronic alcohol misuse. This decreased skeletal muscle mass and function increases risk for frailty and falls, decreases quality of life, and worsens prognosis for other comorbid conditions. After muscle injury or atrophy, satellite cells are activated as myoblasts, which proliferate, differentiate, and fuse with myofibers to aid in repairing and rebuilding skeletal muscle. While decreased myoblast differentiation with ethanol (EtOH) is well-documented, we have also observed fewer myoblasts with in vitro EtOH after the proliferative phase of myogenesis. The extent of this decrease and whether this is due primarily to increased cell death, decreased cell division, or a combination thereof remains unanswered. Therefore, the objective of this study is to quantify effects of EtOH on myoblast cell division and death during the proliferative phase. C2C12 myoblasts were proliferated in the presence or absence of 50 mM EtOH for 24, 48, and 72 hrs. Nuclei were stained with DAPI to quantify differences in cell count at each time point. Lactate dehydrogenase (LDH) activity was measured in cell culture supernatant at 48 and 72 hrs and normalized to cell count to assess overall cytotoxicity. T-tests were used to determine differences between conditions at each time point. There were no significant differences in cell counts between cells grown with 0 and 50 mM EtOH at 24 or 48 hrs. At 72 hrs, EtOH significantly decreased cell count (p=0.03). There were no significant differences for LDH activity at 48 hrs. At 72 hrs, LDH activity was significantly lower in the 50 mM group (p=0.05). These preliminary results suggest that EtOH decreases cell count in a time-dependent manner. Cytotoxicity appeared lower with EtOH, although this is likely due to lower intracellular LDH activity given our previously observed adverse impacts of EtOH on myoblast glycolytic function. Regardless, the present data do not support increased cytotoxicity with 50 mM EtOH. Next steps will be to assess alterations in myoblast apoptosis and cell division with EtOH. Future work based on the results of these experiments will examine possible nutraceutical supplements and exercise-like stimulation as interventions to rescue alcohol-mediated effects on myoblast proliferation, which may improve subsequent differentiation and therefore regenerative capacity.

This work was supported by Texas Tech University (TTU) startup funds and a TTU College of Arts & Sciences Undergraduate Research Experiences grant (DL).

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.
Mitochonic Acid 5 Improves Mitochondrial Respiration Following Alcohol-Induced Mitochondrial Dysfunction
Authors: Donovan Meagan , Mobasheran Parnia , Yang Qinglin
Published: May 2024

Chronic alcohol exposure can lead to the development of Alcohol Induced Cardiomyopathy (ACM). The pathological changes in ACM have been suggested to be largely caused by mitochondrial damage, reduction of ATP, oxidative stress, and apoptosis of the cardiomyocytes. Cardiomyocytes, as cells with high energy demand, rely greatly on mitochondrial homeostasis and ATP production. Thus, therapeutic intervention to specifically target the mitochondria and ATP production can potentially alleviate ACM. Mitochonic acid 5 (MA-5) has been recently identified as a potential therapeutic compound that increases cellular ATP and improves mitochondrial function in several cardiac disorders. Given the potential role of MA-5 in improving mitochondrial function, I speculate that MA-5 could be an effective treatment for alcohol-induced cardiotoxicity. In the present study, I test the hypothesis that MA-5 can mitigate mitochondrial energetics in cultured cardiomyocytes exposed to alcohol. To investigate this, neonatal mouse cardiomyocytes (NMCMs) were isolated from mice at day 1-2 of age and cultured for 6 days with alcohol (50mM). The cells were treated with MA-5 (1μM, 5μM, or 10μM) for 4 hours with serum starvation and then cellular energetic changes were measured using the Mito Stress assay with the Seahorse Bioanalyzer (XF96pro, Agilent). To investigate the impacts of chronic alcohol exposure, rat embryonic myoblasts, H9C2 cells, were treated with 50mM alcohol for 3 weeks. Subsequently, cells were treated with MA-5 (1μM, 5μM, or 10μM) for 24 hours and then attached to the Resipher system (Lucid Lab) for continuous oxygen consumption monitoring and recording. NMCMs treated with 50mM alcohol showed a substantial reduction in basal respiration, maximal respiration, and spare respiratory capacity compared to controls. NMCMs treated with 10μM MA-5 showed a significant improvement in these parameters. Chronically exposed H9C2 cells showed a prolonged decline in OCR. MA-5 treatment improved and maintained oxygen consumption in these cells. In summary, these results demonstrated that alcohol exposed cardiomyocytes experience a decline in cellular energetics that could be corrected by MA-5 treatment. Future in vivo studies on preclinical ACM animal models are warranted to yield further insights into a potential novel therapy for ACM in patients.

This research was supported by grant #T32AA007577 from the NIAAA at the National Institutes of Health.

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.
Nr4a3 Loss Impairs Adipose Mitochondrial Respiration Through Downregulaion of Mek1
Authors: Carter Jared , Tessem Jeffery
Published: May 2024

Type 2 diabetes mellitus (T2D) is a chronic condition affecting nearly half a billion people worldwide. Symptoms of T2D include impaired glucose tolerance, decreased insulin secretion and significant weight gain. While the symptoms of T2D are well-documented, the underlying pathology remains unclear. Recent research has indicated the critical role of the nuclear receptor Nr4a3 in the development of glucose intolerance and weight gain. In individuals with T2D, the Nr4a3 promoter is hypermethylated, leading to decreased Nr4a3 expression. Elucidating the role of Nr4a3 in mitochondrial respiration in adipose will help define the mechanism of T2D onset and treatment. Here we demonstrate the effect of systemic Nr4a3 loss. Full body Nr4a3 KO animals were fed a standard diet. Male, but not female, Nr4a3 KO mice demonstrate impaired glucose and insulin tolerance. Male Nr4a3 KO mice have normal muscle, liver, and kidney respiration rates, while adipose tissue respiration is impaired. The impaired adipose respiration corresponds with increased mass of all adipose depots, increased adipocyte size, and increased lipid droplet size. The analysis of electron transport chain and tricarboxylic acid complex proteins did not reveal significant differences. Instead, the altered respiration is attributed to a decreased Mek1 protein level, and decreased phosphorylation and activation of its downstream target DRP1. Our data demonstrate the critical role of Nr4a3 in adipose tissue through regulating mitochondrial function.

NIH R15DK124835-01A1.

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.
Proteomic analysis of central amygdala systems regulated by the glucocorticoid receptor antagonist mifepristone in the context of alcohol dependence
Authors: Lee Sumin , Romero Laura Monteagudo , Klein Jennifer , Edwards Kimberly , Guidry Jessie , Worthylake David , Edwards Scott
Published: May 2024

Alcohol use disorder (AUD) is a chronic psychiatric disorder characterized by escalated alcohol use and emergence of negative emotional states during withdrawal. Studies show a dysregulation of glucocorticoid signaling in AUD, affecting the hypothalamic-pituitary-adrenal (HPA) axis and the central amygdala (CeA). Excessive activation of glucocorticoid receptors (GRs) is hypothesized to promote cognitive dysfunction, negative emotional states, and escalated drinking. Previous work demonstrates a functional increase in GR activity in the CeA during alcohol withdrawal in alcohol-dependent animal models. Importantly, the GR antagonist mifepristone reduces alcohol-seeking and drinking behaviors in rodents and humans. The aims of this study were to determine differential CeA protein expression in alcohol-dependent rats and examine the effects of mifepristone treatment. We hypothesized that male and female rats will show sex differences in their CeA protein expression and that alcohol-induced reductions in brain anti-stress signaling (e.g., endocannabinoid system) will be normalized by mifepristone treatment. Male and female Wistar rats were exposed to chronic intermittent ethanol vapor (CIEV) or air (control) for 10 weeks. In week 7, half of each group received subcutaneous placebo pellets while the other half received mifepristone pellets (150mg, 21-day slow release). At the end of Week 10, the rats were sacrificed during acute (6-hour) CIEV withdrawal (and identical time in control groups). The CeA was analyzed using proteomics and ingenuity pathway analysis (IPA). In males, a total of 3050 proteins were detected in the CeA. Of these, 142 proteins were altered by alcohol (but not mifepristone), whereas an additional 253 proteins were altered by alcohol and “normalized” by mifepristone. In females, among the 2632 detected proteins, 46 were affected by alcohol (but not mifepristone), and 25 were altered by alcohol and restored by mifepristone. IPA revealed significant changes in canonical pathways, including the endocannabinoid neuronal synapse pathway and oxidative phosphorylation, affected by alcohol but normalized by mifepristone. Future studies will incorporate alcohol self-administration procedures in male and female rats to functionally examine proteins within these pathways as promising “druggable” targets that offer novel therapeutic avenues for AUD.

Supported by NIH-NIAAA R01AA025996, P60AA009803, T32AA007577.

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.
Influence of Individual Characteristics on Critical Environmental Limits in Middle-aged and Older Adults (PSU HEAT Project)
Authors: Leach Olivia , Cottle Rachel , Fisher Kat , Kenney W. Larry , Wolf S. Tony
Published: May 2024

Critical environmental limits represent the temperature/humidity combinations at which heat balance can no longer be maintained for a given metabolic rate. Individual characteristics (e.g., sex, body weight and size, and aerobic fitness) are known to alter environmental heat exchange as well as thermoregulatory effector responses and therefore may modify critical environmental limits. PURPOSE: To determine which individual characteristics, if any, may be associated with higher or lower critical environmental limits [combinations of ambient dry-bulb temperature (Tdb) and relative humidity (rh)] in middle-aged and older adults. Methods: Forty-eight male (n=16) and female (n=32) participants (63±11 yrs; range: 40-92 yrs) were exposed to progressive heat stress at a low metabolic rate designed to reflect activities of daily living. Experiments were conducted in both hot-dry (HD; up to 53°C; ≤25% rh) and warm-humid (WH; ~35°C; ≥50% rh) environments. After determining critical limits for each age group and condition, forward stepwise multiple linear regression analyses were conducted with net metabolic rate (Mnet) and age entered into the model first, followed by sex, body mass, Vȯ2max, body surface area, and LDL cholesterol. Results: After accounting for Mnet and age, both sex and body mass further significantly improved the regression model in the HD environment (R2adj = 0.41, p < 0.001) whereas only sex (R2adj = 0.51, P < 0.001) added significantly to the WH model. Sex explained 11% of the variance in critical environmental limits in HD conditions and 14% of the variance in WH conditions. In both WH and HD environments, females exhibited lower critical combinations of Tdb and rh than their male counterparts. Additionally, body mass was negatively related to critical environmental limits in the HD environments and accounted for 8% of the variance after metabolic rate and age were accounted for. CONCLUSION: These data indicate a modest influence of individual characteristics, specifically sex and body mass, on critical environmental limits in both WH and HD environments in adults between the ages of 40 and 92. SIGNIFICANCE/NOVELTY: These results contrast previously published data in young adults showing no significant contribution of individual characteristics to the upper limits of heat balance at low metabolic rates reflecting activities of daily living. This reflects the heterogeneity of thermal-balance thresholds associated with aging relative to those seen in young adults.

NIH R01 AG067471 (WLK) and NIA Grant T32 AG049676 (RMC).

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.
Adenylate cyclase 6 targets in Mucociliary Clearance
Authors: Arora Kavisha , Arora Yashaswini , Sorensen John , Naren Anjaparavanda
Published: May 2024

Background. Mucociliary clearance (MCC) is a frontline airway defense mechanism that relies on the appropriate interactions between the epithelium, ciliary beat frequency, and the quantity and quality of mucus. MCC is severely impaired in cystic fibrosis (CF) and drives lung function decline and ultimate respiratory failure and death. A great need remains to identify the key molecular players that regulate MCC in CF outside the scope of FDA approved CFTR therapy and to specifically target MCC dysfunction in CF using small molecules. Results. We discover a novel key role of cAMP-synthesizing enzyme Adenylate cyclase 6 (AC6) in MCC via (a) regulation of CFTR chloride channels in the secretory cells that generate airway surface liquid (ASL) component of MCC and (b) AC6 mediated regulation of ciliary beat frequency in the ciliated cells that is required for the mechanical clearance. Specific activators of AC6 (C20: Top candidate) were developed and improved MCC process ex vivo in CF explant tissues and cells. Together these mechanisms and small molecule approach that improve airway fluid balance and cilia function could be beneficial in CF and other respiratory diseases. Experimental Approach. We performed CFTR functional assessment in normal and CF patient derived airway epithelial cells by adapting patch-sequencing approach and using the specific activators of AC6. The effect of AC6 activators on MCC will be evaluated ex vivo in patient derived lung explant tissues , in vivo (micro-CT) in CF rat models and in vitro (ciliary beat frequency and bead flow measurements) in air-liquid interface airway epithelial cultures. Overall, these studies will be mechanistic and translational involving human disease specimens, animal models, drug development and computational analysis. Significance of the results. The proposed research can improve our understanding of epithelial biology and advance activators of AC6 as therapeutic agents to treat mucus clearance defects. These findings can be leveraged to modulate CFTR function and cilia-generate flow, presenting a potential therapeutic avenue for conditions that involve CFTR and cilia dysregulation associated with several respiratory diseases.

National Institutes of Health (NIH) DK080834 and P30-DK117467 and HL147351 (to APN).

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.