bluuebunny/crossref_metadata_2025 huggingface dataset:
Lower Mitochondrial Genome Turnover and Greater Mutation Frequency in Skeletal Muscle of Aged Compared to Adult OKC-HET Rats
Authors: Musci Robert , Fuqua Jordan , Peelor Frederick , Richardson Arlan , Miller Benjamin , Wanagat Jonathan
Published: May 2024

Mitochondrial genomic integrity, which is supported by processes such as replication, repair, and turnover, is critical in supporting metabolism and organismal health. Accumulation of mitochondrial DNA (mtDNA) mutations contributes to mitochondrial and whole-tissue dysfunction, which lead to chronic diseases including sarcopenia. The underlying mechanisms that promote generation and age-related accumulation of mtDNA mutations are unclear. mtDNA mutants may have a replicative advantage over wild-type mtDNA that propagates the accumulation of mtDNA mutations with age. However, there is limited in vivo data regarding effects of age on mtDNA replication, particularly in skeletal muscle. We hypothesized mtDNA synthesis rates would increase with age as mutation burden increased in skeletal muscle in both male and female rats. Methods: We measured mtDNA synthesis over 14 days using the stable isotope tracer deuterium oxide (D2O) and assessed mtDNA copy number and mutation deletion frequency using digital PCR in quadriceps muscle of 9- and 26 month-old (mo) male and female OKC-HET rats, which have heterogenous mtDNA backgrounds. Results: There were no differences in mtDNA copy number between sexes or ages. However, 26 month-od rats had lower rates of mtDNA synthesis compared to 9 month-old rats (9 mo: 0.509 ± 0.009 %/day, 26 month: 0.371 ± 0.048 %/day; p=0.0024) and greater mtDNA half-lives (9 mo: 132 ± 1.79 days, 216 ± 25.63 days; p=0.0009). Concomitant with lower mtDNA synthesis, 26 month-old rats had greater (p=0.003) mtDNA deletion mutation frequency (2.023e-004 ± 7.18e-005) than 9 month-old rats (9.310e-005 ± 2.404e-005). 26 month-old female rats (1.309e-004 ± 2.386e-005) had a lower (p=0.008) mutation burden than male rats (2.74e-004 ± 4.484e-005). Conclusion: Contrary to our hypothesis, mtDNA synthesis declined with age as mutation burden increased in the quadriceps. Because mtDNA copy number was not different between ages, while synthesis decreased, mtDNA turnover declined with age. Altogether, these results suggest lower mtDNA turnover rates may contribute to age-related mtDNA mutation burden. Future studies should test how modulating mtDNA turnover affects mutation frequency and mitochondrial function.

This study was supported by funding from NIH grants R21AG072137 and 5T32AG052363-04.

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 in vivo effects of an ENaC Na+ self inhibition-enhancing mutation
Authors: Nickerson Andrew , Sheng Shaohu , Wang Xueqi , Mutchler Stephanie , Chen Jingxin , Marciszyn Allison , Whelan Sarah
Published: May 2024

Background: Epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron is a critical regulator of extracellular fluid volume and K+ homeostasis. Physiologic concentrations of extracellular Na+ reduce channel activity (open probability; PO) via an allosteric mechanism termed “Na+ self-inhibition,” which involves Na+ ions binding to specific sites in the channel’s extracellular domains. Recent studies have identified human ENaC mutations that alter the Na+ self-inhibition response when assayed in vitro. Such mutations may confer sensitivity or resistance to salt-induced blood pressure changes. Using CRISPR-Cas9 technology, we generated mice carrying a mutation previously shown to enhance Na+ self-inhibition (αH282R) to assess the effects on ENaC-dependent Na+ transport in vivo. We predicted that ENaC activity would be reduced in these mice, leading to enhanced sensitivity to Na+ deprivation or K+ loading, along with accompanying compensatory changes to overcome the predicted loss of function. Methods: We measured urinary electrolyte handling in wild type (WT) and αH282R mice maintained on normal, low Na+, or high K+ diets using metabolic cages. We assessed ENaC activity ex vivo using patch clamp and Ussing chamber methods. We also measured blood chemistry and aldosterone levels in WT and αH282R mice under the dietary conditions described above. Results: When maintained on a normal chow diet (0.3% NaCl), αH2832R mice expressed ENaCs in the distal nephron with reduced PO compared to WT littermates (0.026 vs. 0.067; p<0.01), while ENaC subunit protein expression was unaltered. Amiloride-sensitive short-circuit currents were reduced in the distal colons of αH283R mice versus WT littermates (-12.6 vs. -3.9 μA/cm2; p<0.01). Plasma aldosterone levels were also higher in αH283R versus WT mice on a control diet (610 vs 288 pg/mL; p=0.02), while no other differences in baseline blood chemistry were observed. When challenged with either dietary Na+ restriction (<0.01% NaCl) or K+ loading (10% KCl), WT and αH283R mice responded similarly in terms of body weight maintenance, urinary Na+ and K+ excretion, and systemic electrolyte and metabolic parameters, with the exception of higher aldosterone levels in Na+-restricted αH282R mice compared to WT littermates (2,759 vs. 1271 pg/mL; p<0.01). Conclusions: The αH282R mutation, which enhances Na+ self-inhibition, confers reduced ENaC activity in mice. However, this loss of function may be compensated by ENaC-independent Na+/K+ handling mechanisms and/or increased aldosterone signaling during adaptation to dietary salt stressors.

P30DK079307, T32DK061296.

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.
Circulating Fetal Troponin I May Reflect Cardiomyocyte Proliferation
Authors: Jonker Sonnet , Louey Samantha
Published: May 2024

INTRODUCTION: Circulating biomarkers such as Troponin I (TnI) that are typically indicative of myocardial damage in adults have been reported to be detectable in cord blood of healthy infants delivered both vaginally and by cesarean section prior to labor. We performed serial sampling of healthy fetal sheep from ~20 days before spontaneous birth to 5 days postnatal in order to describe normal developmental values of circulating TnI. METHODS: Seven fetal sheep were surgically instrumented with indwelling catheters advanced into the ascending aorta and the superior vena cava. Following surgical recovery, daily blood samples were taken first thing in the morning into heparin, and the plasma was frozen for later determination of high-sensitivity (hs) TnI levels (BeckmanCoulter UniCel DxI Access IA; log transformed limit of detection =0.30, limit of quantification =0.78, and 99th%ile =1.78). Positive and negative control samples were drawn from an adult ewe during a terminal experimental ligation of the left anterior descending coronary artery and similarly assessed. hs-TnI data were log transformed from ng/L, visually assessed, and outliers removed by the ROUT method. RESULTS: Log(hs-TnI) was 1.47±0.30 at 20 days before birth and declined to 0.88±0.36 in fetuses 12±4 hour before birth (p<0.0001, R2=0.8444). Birth stimulated a delayed, transient peak in hs-TnI (P=0.0058). In the newborn, 43±19 min after birth, levels were 1.39±0.40 (P=0.0650 vs. fetus on day of birth). The day after birth, log(hs-TnI) increased to 2.14±0.63 (P=0.0331 vs. newborn). The second day after birth, levels declined to 1.65±0.48 (P=0.0238 vs. day 1). Adult ewe log(hs-TnI) was below the limit of detection; three hours following coronary artery ligation, levels were 3.21. CONCLUSIONS: This study confirms that hs-TnI levels are moderate to high in healthy sheep fetuses, and reports for the first time that they decline towards term. Why TnI is elevated in the healthy fetus is unknown. If cardiomyocyte TnI is leaked during cytokinesis, the decline towards birth may reflect the diminishing rate of proliferation in this same period. The postnatal hs-TnI peak, above the clinical threshold for evidence of myocardial ischemia in adult humans, may result from hemodynamic, oxidative, or metabolic stresses associated with the establishment of postnatal physiology.

This work was funded by an award from the NHLBI (R01HL142483).

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.
Remodeling of the Human Skeletal Muscle Proteome Found After Long-Term Endurance Training but Not After Strength Training
Authors: Emanuelsson Eric , Arif Muhammad , Reitzner Stefan , Perez Sean , Lindholm Malene , Mardinoglu Adil , Daub Carsten
Published: May 2024

Regular exercise training has tremendous systemic tissue-specific health benefits, but the molecular adaptations to long-term exercise training are not completely understood. The aim of the current study was to investigate the skeletal muscle proteome of highly endurance-trained, strength-trained and untrained individuals and performed exercise- and sex-specific analyses. As a previous investigation from our lab showed limited baseline differences at the gene expression level between highly strength-trained males and untrained males, we hypothesized that the that long-term resistance training results in baseline differences in protein abundances. Additionally, we hypothesized that endurance-trained individuals would have a greater abundance of mitochondrial proteins compared with the untrained and the strength-trained individuals. Liquid-chromatography followed by mass-spectrometry (LC-MS/MS) was performed on skeletal muscle tissue from all individuals. A total of 6713 proteins were detected, with 4269 proteins being expressed in at least 50% of the samples. Over 650 proteins were differentially expressed in the skeletal muscle of endurance-trained individuals compared with controls. Strikingly, 92% of the shared proteins with higher expression in both the male and female endurance groups were known mitochondrial proteins. In contrast to the findings in endurance trained individuals, minimal differences were found in strength-trained individuals and between females and males. Lastly, to determine the preventive-medicine capabilities of long-term training, a co-expression network and comparative literature analysis revealed key proteins and pathways related to the health benefits of exercise, which were primarily related to differences in mitochondrial protein abundance. This network is available as an interactive database resource ( https://inetmodels.com ) where investigators can correlate clinical data with global gene and protein expression data for hypothesis generation. In conclusion, we suggest that long-term endurance training but not strength training results in a r emodeling of the skeletal muscle proteome in humans.

This work was financially supported by grants from the Swedish Research Council (2018-02932), the Swedish Center for Sports Research (2020-D0007, P2022-0082), the National Science Foundation (Award Number: 1951792), the Whitaker International Program and the Karolinska Institute.

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.
Insulin-Mediated Capillary Recruitment Is Absent in the Cutaneous Microvasculature of Women with a History of Gestational Diabetes Mellitus
Authors: Maurer Grace , Hernandez Paola , Schwartz Kelsey , Brustkern Kaila , Jalal Diana , Stanhewicz Anna
Published: May 2024

Women with a history of gestational diabetes mellitus (GDM) have a significantly greater risk of developing cardiovascular disease and type II diabetes compared to women who had an uncomplicated pregnancy (HC). Although the mechanisms underlying this increased risk are unclear, emerging evidence suggests that microvascular endothelial dysfunction, mediated via reduced nitric oxide (NO)-dependent dilation, persists after pregnancy complicated by GDM. Whether this microvascular dysfunction reduces insulin-mediated responses in these women is unexplored. We hypothesized that insulin-mediated capillary recruitment would be attenuated in women with a history of GDM via NO-dependent mechanisms. 24 women (n=12/group; HC: 35±1 years, 24±4 months post-partum; GDM: 33±1 years, 21±3 months postpartum) underwent intradermal microdialysis, which involved the placement of 2 fibers in the ventral forearm for the local delivery of 10−4M insulin alone or 10−4M insulin+15mM NG-nitro-L-arginine methyl ester (L-NAME, NO-synthase inhibitor). The cutaneous blood flow response to post-occlusive reactive hyperemia (PORH; 5 minutes) was assessed before and during insulin perfusion. Red blood cell flux was measured continuously over each microdialysis site by laser-Doppler flowmetry. Cutaneous vascular conductance was calculated (CVC=flux/mean arterial pressure), normalized to maximum (%CVCmax), and presented as PORH area under the curve (AUC, %CVCmax• sec−1). Microvascular capillary recruitment was assessed as ΔAUC (pre-insulin AUC - insulin AUC). Insulin perfusion increased PORH in HC (pre-insulin: 4066±490 vs. insulin: 7848±978 AUC; p=0.03) but had no effect in GDM (pre-insulin: 3765±293 vs. insulin: 6196±779 AUC; p=0.26). Insulin perfusion did not increase PORH at the L-NAME site in either group (p>0.05). Within groups, L-NAME perfusion reduced insulin-mediated capillary recruitment in HC (control: 3781±1109 vs. L-NAME: 470±945 ΔAUC; p=0.04) but not GDM (control: 2431±796 vs. L-NAME: -41±1014 ΔAUC, p=0.11). These data suggest that insulin-mediated capillary recruitment assessed by PORH in the cutaneous circulation is mediated by NO-dependent mechanisms in healthy women with a history of uncomplicated pregnancy and attenuated in healthy women with a history of GDM. This reduction in insulin-mediated microvascular responses may contribute to the increased risk of cardiovascular disease and type II diabetes in these women.

UI Fraternal Order of Eagles Diabetes Research Center Catalyst Grant; NIH HL169201.

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.
Mindfulness-Based Stress Reduction and Autonomic Modulation in Chronic Kidney Disease
Authors: Jeong Jinhee , Zanuzzi Matias , DaCosta Dana , Li Sabrina , Park Jeanie
Published: May 2024

Intro: Chronic kidney disease (CKD) is characterized by chronic overactivation of the sympathetic nervous system (SNS) that leads to increased blood pressure and cardiovascular disease risk. Mindfulness-Based Stress Reduction (MBSR) is an 8-week mindfulness meditation training program that has previously been shown to reduce blood pressure and improve autonomic balance in healthy and some clinical populations; however, MBSR has never previously been tested in CKD and the long-term effects of mindfulness mediation on SNS activity are unknown. Thus, we tested the hypothesis that 8 weeks of MBSR reduces SNS activity and reactivity in CKD patients. Method: In a randomized controlled trial, participants with CKD stages III-IV were randomized to 8 weeks of MBSR (n=17, 64±8 yr, 88% males) or a health education program (HEP, n=12, 63±10 yr, 83% males) that served as the active control intervention. MBSR was comprised of eight 2.5-hour group meetings and a retreat session focused on mindfulness training. HEP was administered in a similar structure and focused on health education. Direct intraneural measures of SNS activity directed to muscle (MSNA) was recorded via microneurography with concomitant beat-to-beat blood pressure and EKG monitoring at rest and during mental stress before and after the 8-week intervention. MSNA reactivity during mental arithmetic-induced stress (3 minutes) was compared between baseline and end of study (EOS) in the MBSR and HEP groups. Results. Baseline characteristics, kidney function, and comorbidities were comparable between groups. There was a significant Group (MBSR vs. HEP) by Time (baseline vs. EOS) interaction in the change in MSNA reactivity to mental stress. The MBSR group had a significant reduction in MSNA reactivity during the 1st, 2nd and 3rd minute of mental arithmetic at EOS (p<0.05 for all), while no change was observed within the HEP group. Resting MSNA and reactivity in blood pressure and heart rate during mental arithmetic remained unchanged in both groups from baseline to EOS. Conclusion. Patients with CKD had an amelioration of sympathetic reactivity during mental stress following 8-weeks of MBSR but not after HEP (control intervention). Our findings demonstrate that mindfulness training is feasible and may have potential beneficial effects on autonomic function in CKD.

NIH R01HL135183; NIH R61AT10457; NIH KL2TR002381; VA Merit I01CX001065.

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.
Peripheral blood mononuclear cells isolated from e-cigarette users have a greater inflammatory cytokine response to LPS stimulation compared to controls
Authors: Wetzel Elizabeth , Cho Josalyn , Stanhewicz Anna
Published: May 2024

Chronically elevated inflammation is a contributing factor in the development and progression of chronic disease, including cardiovascular disease. Biomarker studies suggest that chronic e-cigarette use is associated with elevated circulating inflammatory cytokines and overall vascular inflammation. However, few studies have examined measures of immune cell activity in e-cigarette users. We hypothesized that healthy young adults (18-24 years) who chronically (history of use ≥6 months) and exclusively use e-cigarettes (EC) would have higher plasma concentrations of the inflammatory cytokine TNFα compared to healthy controls (HC), and that peripheral blood mononuclear cells (PBMC) isolated from EC would release more TNFα when stimulated with lipopolysaccharide (LPS) compared to PBMC isolated from HC. 9 HC (20±2 years) and 10 EC (20±1 years; 4±2 years of e-cigarette use) participated in 1 study visit in which we collected 20mL of whole blood via venipuncture and isolated plasma and PBMC. PBMC were incubated in RPMI and 10% FBS with or without 1mg/mL LPS (37°C, 5% CO2). PBMC supernatants were collected at 4 and 24 hours and TNFα concentrations were quantified in plasma and supernatant samples via ELISA. EC had greater plasma TNFα concentrations compared to HC (HC: 1.7±1.5 vs. EC: 3.1±2.7 pg/mL; p=0.03). PBMC supernatants from EC had elevated TNFα following 4 (HC: 28.0±16.9 vs. EC: 52.1±46.5 pg/mL; p=0.04) and 24 (HC: 87.7±71.6 vs. EC: 178.9±121.0 pg/mL; p=0.04) hours of LPS stimulation compared to HC. There were no group of time differences in TNFα in supernatants from unstimulated PBMC (all p>0.05). These data agree with prior findings that chronic e-cigarette use is associated with elevated circulating inflammatory cytokines and suggest that mononuclear cells of e-cigarette users have a greater cytokine response to inflammatory stimuli.

NIEHS/NIH P30 ES005605.

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.
SARS-CoV-2 Decreases Neuronal Activity in Brainstem Respiratory Centers in C57BI6/J Mice
Authors: Restivo Luke , Pasley India , Miller Ryan , Yakoub Abraam , Tseng Chien-Te K , Lazartigues Eric
Published: May 2024

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also called COVID-19, targets the respiratory system, leading to symptoms such as cough, fever, and diffculty breathing. As the mechanisms behind SARS-CoV-2 infections continues to be studied, understanding of how this virus targets specific tissues becomes more apparent. Angiotensin-converting enzyme-2 (ACE2), the target of SARS-CoV infections and a functional receptor for SARS-CoV-2 invasions, is highly expressed in many tissues throughout the body, including the lungs, kidneys, and brain. Notably, our group previously reported that neurons are the main cells within the brain expressing ACE2. Accordingly, we hypothesized that neuronal infection of brainstem respiratory centers by SARS-CoV-2 could contribute to respiratory symptoms associated with COVID-19, specifically respiratory failure. C57BI6/J mice were mock infected or received SARS-CoV-2 (7.5x103 or 1.5x104) intranasally in a BSL-3 facility (University of Texas in Galveston) and were euthanized by CO2 hypoxia after 5 days post infection. The heads were harvested and immersed in formalin for 3 days before being transferred to LSU Health-NO for processing. Brains were carefully dissected out and preserved in optimal cutting temperature (OCT) before sectioning on a cryostat. Sections (30 μm) containing the retrotrapezoid nucleus (RTN) were selected as its neurons are part of the pontomedullary regions that generate the respiratory rhythm and pattern. Immunohistochemistry for c-Fos, a marker of neuronal activity, was used to assess neuronal activity in the RTN. Sections were incubated overnight with a primary monoclonal antibody (rabbit anti c-Fos, 1:1000) followed with a secondary goat antirabbit fluorescent IgG (GFP, 1:200, 1h) antibody. Sections were then mounted to slides in the dark to preserve the fluorescence and observed under the microscope. Images of neurons present in the RTN were captured and neurons were then hand counted and averaged. Neuronal activity in the RTN was decreased with the dose of SARS-CoV-2 used to infect cells in the brain. The greater the dose of SARS-CoV-2, the lower the number of c-Fos-positive neurons was detected in the RTN. Mock brains had the greatest number of neurons and brains infected with 1.5x104 SARS-CoV-2 had the lowest number of neurons in the RTN. This was demonstrated by counting neurons at 3 different intervals and taking the average. We conclude that SARS-CoV-2 targets neurons in respiratory centers, possibly contributing to impaired respiratory function in infected patients.

NIH/NHLBI HL163814.

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.
Resident macrophages in kidney medulla are critical in cleaning intratubular sedimentary particles
Authors: Shen Xiao (Gregory) , he jian , Shi Peng
Published: May 2024

During the passage of the glomerulus filtrate through the renal tubular system, various microscopic sedimentary particles, including mineral crystals resulting from urine concentration, are generated. Dislodging these particles in the intratubular compartment is critical to ensure free flow of filtrate and the final formation of urine. However, it is generally assumed that particle removal is solely attributed to urine flush and whether there is other devoted mechanism is unknown. In this study, we uncovered that most macrophages in the kidney medulla of mice constitutively formed transepithelial protrusions and were “sampling” urine contents, as revealed by high-resolution confocal microscopy. This phenomenon was less pronounced in the cortex. The transepithelial protrusions of medulla macrophages were formed in a transcellular route instead of between epithelial cells, so the overall junctional architecture of tubular epithelium was intact. Juxtatubular macrophages were effcient in sequestering and phagocytosing intraluminal particles through their transepithelial protrusions, and would make transmigration to the tubule lumen to "escort" the excretion of sediment particles, as documented by live two-photon microscopy recording. In accordance, abundant particle-carrying macrophages could be detected in the urine. As such, specifically depleting renal macrophages precipitated kidney stone formation in a hyperoxaluria model. Moreover, naïve mice with underrepresentation of renal macrophages were prone to developing various intratubular sediments. RNA-seq analyses unveiled that medulla macrophages were enriched with genes associated with cell adhesion relative to cortex macrophages. Mechanistically, we found that integrin β1-mediated ligation to the tubular epithelium was crucial to the transepithelial behaviors of medulla macrophages, as evidenced by both pharmacological and genetic approaches. This study unveils a previously unappreciated role of renal medulla macrophages in keeping the tubular system unobstructed by removing intratubular particles. These findings may pave way for developing novel therapeutics for nephrolithiasis.

National Natural Science Foundation of China (32170894, 31970898 and 82170441) and the Natural Science Foundation of Zhejiang Province (LZ22C080001).

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.
Streptococcus pneumoniae infection promotes the long-term development of an atherosclerotic phenotype in atheroprone ApoE-deficient mice
Authors: Peters Lisa , Simmons Szandor , Ginsbach Laura F. , Ramezani Rad Pegah , Brack Markus C. , Nageswaran Vanasa , Behrendt Ulrike
Published: May 2024

Background: Epidemiological studies have identified pneumonia to be closely linked to an increased risk for cardiovascular events. Potential causality of this association, however, remains to be established, and underlying mechanisms are yet to be resolved. Here, we tested the hypothesis that prior pulmonary infection with Streptococcus pneumoniae accelerates the development of an atherosclerotic phenotype in mice. Such a phenotype would explain the increased cardiovascular risk in the epidemiological data. Methods: Atheroprone ApoE−/− mice were fed a high fat diet, and after 6 weeks nasally infected with either 2.5 x 107 S. pneumoniae or vehicle control. Antibiotic therapy was initiated 60h post infection (p.i.) by s.c. injections of ampicillin on 5 consecutive days followed by oral administration of amoxicillin for 10 days. At days 35 or 90 post-infection, aortas were harvested and assessed for vasoreactivity and arterial stiffness by wire myography. Morphological changes of the aortic vessel wall and eNOS expression levels were quantified by western blot and histological stainings. Results: Unexpectedly, 35 days p.i. aortas of infected mice presented with decreased aortic stiffness, increased endothelial-independent vasorelaxation and decreased wall thickness due to lower collagen-1 expression and reduced elastin fragmentation when compared to sham animals. After 90 days, however, these effects had reversed and animals with prior pneumonia showed increased aortic stiffness, attenuated endothelial dependent vasorelaxation that was associated with reduced eNOS protein expression and phosphorylation, and increased atherosclerotic plaque burden relative to vehicle control. Conclusion: Single episodes of pneumonia promote a delayed pro-atherogenic phenotype that manifests as endothelial dysfunction, aortic stiffening and enhanced plaque burden. Underlying mechanisms are currently under investigation to identify risk predictors for patient stratification and clinical targets for prevention of cardiovascular disease following pneumonia.

Funding: German Ministry of Education and Research (BMBF) in the framework of SYMPATH (01ZX1906A).

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.
Elucidation of the Molecular Signal Linking Psychological Trauma to T-lymphocyte Inflammation
Authors: Natour Tamara , Lauten Tatlock H. , Reed Emily C. , Case Adam J.
Published: May 2024

Post-traumatic stress disorder (PTSD) is a psychiatric disorder that not only exhibits significant behavioral pathology, but also increases the likelihood of comorbid inflammatory conditions such as autoimmune and cardiovascular diseases. While PTSD patients experience heightened systemic inflammation, the exact link between psychological trauma and immune dysregulation remains to be elucidated. Our group has recapitulated the psychological trauma-induced inflammatory phenotype in a pre-clinical murine model of PTSD known as repeated social defeat stress (RSDS). Importantly, a key site of this inflammation appears to originate from T-lymphocytes in the spleen, and targeted splenic nerve denervation was able to completely prevent splenic T-lymphocyte inflammation after RSDS (p<0.0001). This critical observation suggests the T-lymphocyte response to psychological trauma is neural derived as opposed to circulating, therefore, we began systematic investigation into possible neural-derived signals that may lead to enhanced T-lymphocyte inflammation. To investigate whether the signal involves the T-cell receptor (TCR), we utilized OT-I and OT-II mice that possess non-mouse antigen-restricted TCRs. RSDS induced T-lymphocyte inflammation in these mice similar to wild-type, therefore, suggesting the signal is not antigenic in nature (p>0.05). Next, we examined the role of norepinephrine and adrenergic signaling using both pharmacological blockade and genetic knockouts of both alpha and beta adrenergic receptors, but this too was insuffcient to reverse the RSDS-induced T-lymphocyte inflammation (p>0.05). Following this, we examined the role of damage associated molecular patterns (DAMPs) by pharmacological blockade of the toll-like receptor 4 (TLR4), yet again, this intervention did not attenuate T-lymphocyte inflammation after RSDS (p>0.05). Last, our data suggests interleukin 6 (IL-6) is upregulated early after psychological trauma; therefore, we examined the role for cytokine-induced T-lymphocyte inflammation. Using anti-IL-6 antibodies during RSDS, T-lymphocyte inflammation appeared blunted compared to wild-type animals (p<0.05). Moreover, T-lymphocytes treated with only IL-6 expressed similar inflammation gene profiles as those from RSDS animals (p<0.05). Taken together, our data suggest IL-6 may be the T-lymphocyte inflammation-inducing signal, but additional studies are warranted to understand the exact source of IL-6 and mechanisms underlying IL-6’s ability to drive T-lymphocyte inflammation.

R01HL158521.

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.
Machine learning techniques for predicting microvascular network hemodynamics
Authors: Ebrahimi Saman , Bagchi Prosenjit
Published: May 2024

Existing techniques to image microvascular networks in vivo do not allow a direct and accurate measurement of several hemodynamic variables such as the wall shear stress (WSS). They cannot also provide a full 3D characterization of blood velocity and red blood cell (RBC) concentration profiles in every vessel in a microvascular network. Such physiological parameters however play critical roles in blood flow regulation, disease progression, angiogenesis, and hemostasis. Theoretical models of network blood flow, often used for hemodynamic predictions in experimentally acquired images of the microvascular network, cannot provide the full 3D blood velocity, RBC concentration and WSS profiles as these models assume one-dimensional flow governed by Poiseuille’s law. In contrast, high-fidelity computational models that simulate flow of blood as a suspension of deformable RBCs and model the 3D vessel geometry can readily predict such physiological variables with high accuracy. Such high-fidelity computational models have recently been applied to predict hemodynamic variables in microvascular networks in silico composed of many vessels and vascular junctions. Such models, however, tend to become computationally expensive with increasing size of the network; therefore, they are not feasible for use in large networks at organ scale. To overcome this limitation, we developed a machine learning (ML) approach that can predict hemodynamic variables in 3D and with high accuracy. The ML models are based on artificial neural network and convolution-based U-net models. Using experimentally acquired images of the microvascular networks, inputs at a few inlets, and a “bank” of database that we have developed, these ML models can predict flow and RBC distribution in all vessels in the network, 3D blood velocity and RBC concentration profiles over the cross-section of every vessel, 3D WSS profiles, and time-dependent variations in blood flow in any vessel. The prediction from the ML models compare very well against the true data. Also, the prediction time using ML is significantly less than that of the high-fidelity models. The current ML models, which to our knowledge are the first of their kind, are highly promising for image-based predictions of sub-cellular resolved capillary hemodynamics in organ-scale networks. This study paves the way for AI to make hemodynamic predictions in organ-scale capillary vessel networks while retaining the sub-cellular scale details and overcoming the limitations of the in vivo imaging techniques, with potential applications in hematological and microvascular disorders, angiogenesis, and vascular-mediated drug delivery.

Funded by NIH (R01-EY033003) and NSF (CBET 2302212).

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 ERK and NF-KB pathway in neurally mediated enhanced expression and traffcking of SGLT2 in congestive heart failure
Authors: Patel Tapan , Zheng Hong , Patel Kaushik
Published: May 2024

Congestive heart failure (CHF) is characterized by the activation of neurohumoral systems concomitant with avid fluid retention. Recently we showed enhanced expression of sodium-glucose cotransporter 2 (SGLT2) in rats with CHF. However, the exact molecular mechanism linking enhanced sympathetic tone and enhanced expression and translocation of SGLT2 in CHF is not clear. The objective of this study was to investigate the role of enhanced sympathetic tone on the expression and traffcking of SGLT2 via activation of protein kinase A (PKA), extracellular signal-regulated kinases (ERK), and nuclear factor kappa B (NF-KB) in the renal cortex of CHF rats. Male Sprague Dawley rats were subjected to either the left coronary artery ligation for the CHF group or Sham surgery. Four weeks after surgery, expression of protein levels (PKA, ERK and NF-KB) were assessed by Western blotting, whereas the location of SGLT2 expression was determined by immunohistological and immunofluorescent staining in the cortical region of kidneys from Sham and CHF rats. Effect of exogenous norepinephrine (NE) (10μM for 24hrs) on expression and traffcking of SGLT2, phosphorylation of ERK and NF-KB in HK2 cells were examined in an in vitro model. Expression of SGLT2 was significantly enhanced in the renal cortex of rats with CHF versus Sham (0.83±0.06 vs. 0.58±0.02). Also, the translocation of SGLT2 was significantly enhanced towards the membrane in CHF rats (0.92±0.11 vs. 0.33±0.07). Phosphorylation of PKA (0.05±0.01 vs. 0.01±0.01), ERK (0.32±0.02 vs. 0.23±0.02) and NF-KB (0.71±0.14 vs. 0.08±0.06) were significantly enhanced in CHF compared to Sham. NE treatment significantly increased expression of SGLT2 (0.26±0.03 vs. 0.15±0.02), traffcking of SGLT2 (0.45 ± 0.09 vs. 0.15 ± 0.03) towards membrane, phosphorylation of ERK (0.38±0.05 vs. 0.14±0.02) and NF-KB (0.61±0.08 vs. 0.17±0.07) in HK2 cells. Thus, NE treatment enhanced the phosphorylation of ERK, NF-kB as well as traffcking of SGLT2. Immunohistological and immunofluorescent staining demonstrated increased expression of SGLT2 in the kidneys of rats with CHF. Similar results were observed in immunofluorescent staining of human kidney cells treated with NE. In conclusion, these findings indicate that NE activates ERK and NF-KB pathways which leads to enhanced traffcking and overexpression of SGLT2 in CHF. These results provide a molecular link between enhanced sympathetic tone, ERK and NF-KB pathway and the traffcking of SGLT2 to the luminal membrane of renal proximal tubular cells and consequent increase in sodium retention in rats with CHF.

This study was funded by NIH R01-DK-129311.

This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.
The role of PPP1R3G in promoting necroptosis in ischemia reperfusion induced acute kidney injury
Authors: Tran Minh , Parris Colby , Wang Lei
Published: May 2024

Recent discoveries have revealed necroptosis as a major contributor to the pathogenesis of acute kidney injury (AKI). Protein phosphatase 1 regulatory subunit 3G (PPP1R3G) has been linked to the essentiality of RIPK1-dependent apoptosis and type I necroptosis. Nonetheless, the involvement of PPP1R3G in the regulation of necroptosis during ischemia reperfusion-induced acute kidney injury (IR-AKI) has yet to be elucidated. Here, we investigated the role of PPP1R3G in the regulation of necroptosis in IR using an in vitro cellular I/R model and in vivo IRI-AKI mouse model. Primary proximal tubular cells (PTCs) from C57BL/6 and Ppp1r3g−/− mice were cultured and cell I/R was induced through hypoxia/reoxygenation. Cell viability and necroptosis were analyzed using CellTiter-Glo assay and Annexin V-Cy3/ SYTOX green staining, respectively. The expression levels of necroptosis, oxidative stress, and inflammation factors were determined by real-time PCR, western blotting, immunofluorescence staining, and ELISA. In the in vivo IRI-AKI model, bilateral renal pedicles clamping was conducted in both C57BL/6 and Ppp1r3g−/− mice. The kidney injury and function were evaluated by plasma creatine, BUN, and KIM-1 levels and GFR values, respectively. Kidney morphological evidence of injury was assessed by PAS staining and cell death by immunofluorescent staining. We found that knockout of PPP1R3G in the primary tubular cells significantly preserved cell viability and attenuated tubular cell death. This protective effect was associated with the reduced expression and phosphorylation of necroptosis factors (RIP1, RIP3, and MLKL), and inflammatory factors (IL-6, IL-10 and TNF-α) in I/R injury cells. Deletion of PPP1R3G attenuated the I/R induced kidney injury indicated by 54% decrease in the plasma creatine (2.28±0.12mg/dL vs. 1.09±0.07mg/dL, p<0.0001, n=7) and 45% higher GFR values than WT mice at day 3 after reperfusion. Histology showed less diffuse renal tubular necrosis and casts comprising necrotic cells and debris in Ppp1r3g−/− mice, as well as 60% less cell death compared to WT mice. Moreover, the application of chemical compounds to prevent inhibitory phosphorylation of RIPK1 significantly reinstated cell death in Ppp1r3g−/− cells. In conclusion, our findings demonstrate, for the first time, that Ppp1r3g-mediated necroptosis significantly promotes IRI-AKI. Therefore, targeting Ppp1r3g to reduce necroptosis in the kidney holds promising potential for significant clinical benefits.

National Institutes of Health Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL.

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.
Breathing 5% carbon dioxide for 1 minute increases cerebral blood flow during lower body negative pressure
Authors: Harrell John , Beri Rachel , Edelsztein Lail , Muriithi Andrew , Pollock Ross
Published: May 2024

Objective: Lower body negative pressure (LBNP) is an established surrogate of hypergravity to investigate responses to central hypovolemia, including the reduction in cerebral blood flow. Increased inspired carbon dioxide (CO2) causes cerebrovascular dilation and increases cerebral blood flow. We aimed to determine whether breathing 5% CO2 for 1 minute increased cerebral blood flow during LBNP. We hypothesized 5% inspired CO2 would increase cerebral blood flow during LBNP. Methods: We collected data from 15 (9 M/6 F, 31±8 yr, 25.6±3.2 kg/m2) healthy individuals. Subjects breathed room air or a mix of 5% CO2, 21% oxygen, balance nitrogen gas for 1 minute prior to initiation of 2 minutes of -60 mmHg LBNP and throughout the duration of LBNP. We allowed a 2-minute wash-out between trials. We measured middle cerebral artery velocity (MCAv) with transcranial Doppler ultrasound and continuous, non-invasive blood pressure (MAP) with a finger plethysmograph. We corrected MCAv for MAP and present it as cerebrovascular conductance index (CVCi). We also measure ventilation and gas exchange with a pneumotachometer and gas analyzer. Results: All measured variables were similar prior to the initiation of room air and 5% CO2 prebreathe ( p > 0.05). Inspired (1±1 vs. 36±1 mmHg) and end-tidal (38±4 vs. 47±3 mmHg) CO2 were greater during the 5% inspired CO2 trial ( p < 0.05, main effect of gas). LBNP caused an increase in heart rate, decrease in stroke volume, decrease in cardiac output, and an increase in total peripheral resistance ( p < 0.05, main effect of LBNP). These variables were similar regardless of inspiratory gas. MCAv (40±16 vs. 53±19 cm/s), MAP (94±10 vs. 99±11 mmHg), and CVCi (0.46±0.22 vs 0.56±0.24 cm/s/mmHg) were all greater during the 5% inspired CO2 trials ( p < 0.05, main effect of gas). Discussion: Breathing a mixture of 5% CO2 for 1 minute increased cerebral blood flow and blood pressure during a mimic of hypergravity stress. Despite technical challenges of integration into high-performance aircraft, increasing inspired CO2 could contribute to better tolerance to hypergravity stress.

Funding was provided by the Air Force Offce of Scientific Research and King’s College London.

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.
Epigenetic changes to the carotid body drive hypertension in perinatal nicotine exposed rats
Authors: Zhu Fengli , Liu Jie , Rehan Virender , Jendzjowsky Nicholas
Published: May 2024

Background: Epigenetic changes can be shaped by a wide array of environmental cues as well as maternal health and behaviors. One of the most detrimental behaviors to the developing fetus is nicotine exposure. Perinatal nicotine exposure remains as a significant risk factor for cardiovascular health and in particular, hypertension, as it has been shown to alter angiotensin signaling. Hypothesis: We hypothesized that perinatal nicotine exposure alters carotid body angiotensin signaling to induce hypertension in adult life. Methods: Pregnant Sprague-Dawley rats were exposed to nicotine (2mg/kg SQ, or PBS- control) from estrous day 6 to post-natal day (PND) 21. Experiments on pups took place from PND 56-90. Carotid bodies were harvested pooled together for RNAseq and confirmation of unbiased transcriptomics was conducted with qPCR. Blood pressure was measured with indwelling telemetry. ELISA assessed serum angiotensin. PC12 cells were used a surrogate for carotid body glomus cells and were used for calcium imaging. Results: Unbiased RNAseq of carotid bodies from nicotine exposed pups compared to saline control pups showed that KEGG pathways with significant angiotensin signaling components including Neuroactive ligand-receptor interaction (Log2FC=2.4, p=7.5x10−8) and Dopaminergic synapse (Log2FC=2.3, p=0.0007) were significantly upregulated. qPCR of carotid bodies demonstrated that Angiotensin II type 1 receptor (56%↑), Angiotensin II type 2 receptor (15%↑) and TRPV1 channels (63%↑) were upregulated in nicotine exposed pups compared to controls. Blood pressure measured in nicotine exposed pups was suppressed 1 day after bilateral carotid body denervation. Circulating angiotensin assessed from serum was similar between nicotine exposed and control pups. Calcium imaging of PC12 cells showed that nicotine significantly increased spike frequency in response to nicotine (0.2mg/ml). Incubation with nicotine + Angiotensin (50ng/ml) did not further increase spike frequency. This increased excitation was suppressed with losartan (3μM), AMG9810 (TRPV1 antagonist, 10μM) and combined blockade (p<0.05). Conclusion: Our data demonstrate that adverse maternal behaviors produce epigenetic changes to carotid bodies of offspring resulting in neurogenic hypertension. The epigenetic changes involve an Angiotensin-Angiotensin II type 1 receptor-TRPV1 channel axis in the carotid body without any significant systemic changes. We suggest carotid body modulation may have a significant health impact on offspring with adverse perinatal environmental exposures.

UCOP TRDRP T32IP4707, T32KT4708.

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.