bluuebunny/crossref_metadata_2025 huggingface dataset:
High Glucose limits Autophagy via inhibition of Nuclear Translocation of Transcription Factor EB
Authors: Kramer Benjamin , Kobayashi Satoru , Gerdes Martin , Liang Qiangrong
Published: November 2023

Cardiovascular disease is the major consequence resulting in morbidity and mortality amongst diabetic patients. Hyperglycemia is a known independent risk factor associated with diabetic heart failure. However, the mechanisms that mediate hyperglycemia‐induced cardiac damage remain poorly understood. Previous literatures show that generation of reactive oxygen species (ROS) and lack of cell maintenance compromise cellular sustainability. Autophagy is the principle pathway that maintains cellular homeostasis via lysosomal degradation of proteins and organelles. We previously showed that the inhibition of autophagy is a protective response in hyperglycemia‐induced cardiac injury. Transcription Factor EB (TFEB), known as a master regulator of autophagy‐lysosome pathway, controls the expression of autophagy‐related proteins and lysosomal proteins. In the present study, we determined if high levels of glucose could affect the expression and the nuclear localization of TFEB, and thus autophagic activity, in cultured cardiac cells. Western blotting results suggest that the total protein expression level of TFEB remains consistent under both high (30mM) and physiologic (5.5mM) glucose conditions. However, the nuclear localization of TFEB is markedly reduced in cardiac myocytes cultured under high glucose as shown by immunofluorescence staining and confocal microscopy, indicating that high glucose inhibits the nuclear translocation of TFEB. In addition, the inhibition of TFEB nuclear localization is accompanied by a reduction of lysosomal associated membrane protein 1 (LAMP‐1), suggesting a reduced autophagy‐lysosome activity. Indeed, we show that high glucose inhibits autophagy and induces cell death. However, restoration of autophagic activity by Rapamycin, a known inducer of autophagy, exacerbates high glucose‐induced cell death, suggesting that the inhibition of autophagy is an adaptive response that protects against high glucose toxicity. Interestingly, Rapamycin treatment increases the nuclear translocation of TFEB as shown by increased co‐localization of TFEB and DAPI, a DNA dye that stains nuclei. Rapamycin also reverses the expression level of LAMP‐1. Collectively, our findings suggest that high glucose conditions suppress the nuclear translocation of TFEB, which may be responsible for the protective role of inhibited autophagy in hyperglycemia‐induced cardiac injury. Future studies are warranted to investigate the molecular mechanism that inhibits nuclear translocation of TFEB in high glucose and to determine whether TFEB overexpression or knockdown can regulate autophagic activity, thereby affecting hyperglycemia‐induced cardiac injury.
Late Na+ Current Contributes to Arrhythmogenic Diastolic Ca2+ release in catecholaminergic polymorphic ventricular tachycardia (CPVT)
Authors: Radwanski Przemyslaw , Ho Hsiang‐Ting , Knollmann Björn , Belevych Andriy , Gyorke Sandor
Published: November 2023

Arrhythmias caused by abnormal impulse formation including CPVT are associated with aberrant diastolic Ca2+ release (DCR) from the sarcoplasmic reticulum. Despite high response of CPVT to agents directly affecting Ca2+ cycling, the incidence of refractory cases is still significant. Surprisingly, these patients often respond to treatment with Na+ channel blockers. However, the relationship between Na+ influx and the arrhythmogenic disturbances in Ca2+ handling (aberrant Na+/Ca2+ signaling) in CPVT remains elusive. To address this issue we used a murine model of cardiac calsequestrin‐associated CPVT. We performed confocal microscopy in isolated ventricular myocytes to assess Ca2+ handling during various pharmacological interventions. Immunocytochemistry experiments were performed to determine the structural underpinnings of Na+/Ca2+ signaling. Late Na+ current (INa) was assessed in whole cell patch‐clamp mode. To monitor arrhythmic activity in vivo surface electrocardiograms were performed before and after intraperitoneal injection of epinephrine (1.5mg/kg) and caffeine (120mg/kg). Immunocytochemistry experiments revealed that neuronal Na+ channels (nNav) colocalize with the ryanodine receptors (RyR2) Ca2+ release channels on the sarcoplasmic reticulum. Isoproterenol (Iso; 100nM) induced late INa in isolated CPVT myocytes which was sensitive to 100nM tetrodotoxin (TTX). Furthermore, nNav blockade with either TTX, riluzole (10μM) or flecainide (2.5 μM) reduced DCR frequency. This antiarrhythmic effect on cellular level translated in decreased ventricular tachycardia (VT) incidence in vivo. On the other hand, nNav augmentation with β‐Pompilidotoxin (β‐PMTX, 40μM) increased both the late INa as well as DCR frequency and VT all of which were reversed by nNav blockade with riluzole or flecainide. These data suggest that Iso‐promoted, nNav‐mediated late INa contributes to the generation of arrhythmogenic DCR in CPVT and can therefore serve as an antiarrhythmic target.

Support or Funding Information

This work was supported by NIH Grants R01‐HL074045, R01‐HL063043 (to S.G.) and K99‐HL127299 (to P.B.R).
Verapamil But Not Lidocaine Suppresses Ischemia‐Induced Ventricular Fibrillation in The Isolated Female Rat Heart
Authors: Davitt Katlin , Hensley Lauren , Clements‐Jewery Hugh
Published: November 2023

Background and Goals

Ventricular fibrillation (VF) triggered by myocardial ischemia is believed to account for the majority of sudden cardiac death, which is responsible for over 300,000 deaths annually in the United States, indicating the need for effective and safe pharmacological therapeutic prevention. Historically, animal models used to identify antiarrhythmic drugs have excluded female models of ischemia‐induced VF, raising the possibility that the paucity of effective and safe antiarrhythmic drugs available clinically is in part due to drugs lacking efficacy in females. In the present study we evaluated in isolated female rat hearts the antiarrhythmic efficacy of verapamil and lidocaine at concentrations that are known to be antiarrhythmic in male rat hearts.

Methods

Hearts were isolated from male and female rats and perfused in the Langendorff mode with modified Krebs solution containing 3mM K+. Six different treatment groups were used (n=12–13 per group). There were corresponding separate male and female groups: control (vehicle ‐ water); verapamil (300nM) and lidocaine (12.93μM). After a 5 min baseline perfusion period the solution was switched in a randomized and blinded manner to one of the test solutions. After a further 15 minutes, regional ischemia was induced by left main coronary ligation and maintained for 30 min. The ECG was used to identify the occurrence of VF and other arrhythmias during ischemia as well as for the measurement of electrocardiographic intervals. An intraventricular balloon was used to record left ventricular developed pressure (LVDP) and end diastolic pressure. Coronary flow was recorded digitally using the calibrated signal from a pump controller.

Results

Verapamil and lidocaine produced near identical hemodynamic effects in male and female hearts consistent with their established mechanisms of action. Compared to control hearts, verapamil decreased LVDP but increased coronary flow whereas lidocaine decreased both heart rate and coronary flow. The VF incidence was 54% and 45% in control male and female hearts respectively, which was lower than that observed in previous studies from our laboratory. The incidence of VF in the presence of verapamil was similar in male and female hearts (17% & 8% respectively) but lidocaine suppressed VF in male hearts only (8% VF vs 46% VF in females).

Conclusions

At concentrations that are antiarrhythmic in male rat hearts, verapamil but not lidocaine suppressed VF in female rat hearts. Further investigation is merited.

Support or Funding Information

Supported by an intramural grant from the West Virginia School of Osteopathic Medicine.
Connexin hemichannels and pannexin channels contributes to the intracellular Ca2+ signal activated by PAF in endothelial cells of mesenteric post‐capillary venules
Authors: Burboa Pia C , Poblete Ines , Figueroa Xavier
Published: November 2023

Endothelial cells form a permeability barrier to macromolecules that can be modulated in post‐capillary venules through the activation of Ca2+‐dependent signaling pathways by pro‐inflammatory agents, such as platelet‐activating factor (PAF). This Ca2+ signaling depends on Ca2+ release from the endoplasmic reticulum and Ca2+ influx from the extracellular space, in which may be involved membrane channels formed by connexins (i.e. hemichannels) or pannexins. We analyzed the participation of these channels in the PAF‐elicited intracellular Ca2+ concentration ([Ca2+]i) increase. To this end, we used the intact mesenteric vascular bed and primary cultures of mesenteric endothelial cells (EC) of resistance arteries (EC‐A) and venules (EC‐V). Changes in [Ca2+]i were recorded using the fluorescent Ca2+ indicator Fluo‐4 and activity of connexin hemichannels or pannexin channels was evaluated by assessing ethidium uptake in EC and intact vessels. As expected, 1 μM acetylcholine induced an increase of [Ca2+]i in EC‐A, but not in EC‐V and, in contrast, 10 nM PAF evoked a response only in EC‐V. ATP (5 μM) activated a Ca2+ response in both EC‐A and EC‐V. In addition to activate a Ca2+ signal in EC‐V, PAF also induced an increase in ethidium uptake in these cells and in venules of intact mesenteric bed, without affecting the ethidium signal in mesenteric arteries. Treatment for 15 min with the connexin blocking peptide 37, 43Gap27 or the pannexin‐1 blocking peptide 10Panx abolished the increase in [Ca2+]i and ethidium uptake induced by PAF, suggesting that connexin hemichannels and pannexin‐1 channels contribute to the PAF‐activated Ca2+ signal. Cx37, Cx40, Cx43 and pannexin‐1 were found to be expressed in intact venules, intact resistance arteries and in EC‐V and EC‐A by immunofluorescence analysis and western blot. Connexin and pannexin channels are permeable to Ca2+, but they may also trigger Ca2+ signals through ATP release and, consistent with this, blockade of purinergic receptors with PPADS blunted the PAF‐induced [Ca2+]i increase. These results suggest that the intracellular Ca2+ signaling activated by PAF in endothelial cells of post‐capillary venules is mainly mediated by ATP release through connexin hemichannels and/or pannexin‐1 channels, which, in turn, leads to activation of purinergic receptors. However, direct Ca2+ influx via connexin hemichannels or pannexin‐1 channels may also contribute to the response

Support or Funding Information

FONDECYT 1150530
Dual and Divergent Location‐Specific Functions of Active Caspase 3 are Regulated by MK2
Authors: Damarla Mahendra , Carino Kathleen , Suresh Karthik , Johnston Laura , Kim Bo , Kolb Todd , Damico Rachel , Machamer Carolyn , Shimoda Larissa , Hassoun Paul
Published: November 2023

Background

We have previously shown that MK2 deficiency leads to cytoplasmic sequestration of active caspase 3, prevention of apoptosis and is associated with a preserved endothelial barrier function in response to endotoxin challenge. While caspase 3 is widely described as the executioner of apoptosis, it has numerous cytosolic substrates that are not related to execution of apoptosis. Therefore, we hypothesized that cytosolic caspase 3 promotes endothelial barrier function and MK2 converts caspase 3's barrier protective function to apoptosis‐promoting thereby stimulating pulmonary vascular permeability.

Methods

Human lung micro‐vascular endothelial cells (HMVECs) were exposed to a non‐apoptotic, thrombin, or an apoptotic, lipopolysaccharide (LPS), stimulus and endothelial barrier function was assessed using electric cell‐substrate impedance sensing (ECIS). After exposures, a subset of HMVECs were evaluated for actin cytoskeletal rearrangement using phalloidin staining. In addition, HMVEC cell lysates were harvested for protein analyses. Caspase inhibition was achieved using the pharmacological inhibitor q‐VD‐OPH.

Results

Exposure of HMVECs to LPS led to an initial enhancement of endothelial barrier integrity as measured by an increase in electrical resistance, followed by pronounced endothelial barrier disruption which did not recover. Intriguingly, the LPS‐induced transition from barrier enhancement to endothelial barrier disruption is associated with nuclear translocation of active caspase 3 and subsequent apoptosis. Knockdown of MK2 prevented LPS‐induced nuclear translocation of caspase 3 and apoptosis. In response to thrombin, HMVECs demonstrate a dose dependent transient decrease in endothelial barrier function as measured by a drop in electrical resistance followed by a rapid recovery on ECIS. Inhibition of caspases in HMVECs led to a similar drop in thrombin‐induced endothelial barrier function but notably there was no subsequent barrier recovery; lack of barrier recovery was not a result of endothelial cell death. Interestingly, LPS but not thrombin leads to MK2 activation.

Conclusion

Taken together, our results suggest that MK2 phosphorylates caspase 3 leading to its nuclear translocation, subsequent apoptosis and barrier disruption; whereas unphosphorylated active caspase 3 remains cytosolic and promotes endothelial barrier integrity.

Support or Funding Information

NIH‐ NHLBI
Smooth Muscle Cell and Endothelial Cell Expression Profiles for Membrane Receptors: Effects of Aging and Vessel Type
Authors: Boerman Erika M , Shaw Rebecca , Ramnath Anjana , Joshi Trupti , Segal Steven S
Published: November 2023

Smooth muscle cells (SMCs) and endothelial cells (ECs) interact to control dilation and constriction of resistance arteries, thereby regulating tissue blood flow and oxygen delivery. The functions of and interactions between SMCs and ECs are regulated by multiple membrane receptors and ion channels. While the expression of genes encoding these proteins varies between cell types, how such expression is affected by aging and may vary between vascular beds are poorly understood. To probe these relationships, we applied genomics and informatics to test the hypothesis that the expression profile of membrane receptors mediating vascular reactivity to physiological agonists varies not only with cell type, but is affected by biological age and vessel type. Individual mesenteric artery (MA) and superior epigastric artery (SEA) segments were collected from Old (24–26 months) and Young (3–6 months) male C57BL/6 mice; each vessel was dissociated to obtain ~5,000 EC and 500 SMCs (n=5 per group). Total RNA was extracted from each cell sample; custom Taqman qRT‐PCR arrays were used to determine the expression profile of receptors. Critical threshold (ΔΔCT) values were generated and t‐tests computed for identifying significant changes at P<0.05. Differences in expression levels of genes for > 25 receptors were identified across cell types, ages, and arteries. Heat maps and distance matrices illustrate up‐ and down‐regulation of receptors for respective comparisons. The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was applied to predict downstream function and signaling pathways, to construct networks of co‐expression and predict protein interactions. Our findings illustrate pronounced differences in expression profiles between ECs and SMCs while revealing more subtle differences with aging and between vessel types.

Support or Funding Information

NIH R37HL041026 & F32HL118836
Cardiac Stretch‐Induced Transcriptomic Changes Are Direction Dependent
Authors: Buchholz Kyle S. , Tan Philip , Zambon Alexander C. , Saucerman Jeffrey J. , McCulloch Andrew D. , Omens Jeffrey H.
Published: November 2023

The goal of this study was to investigate the effect of the major axis of biaxial mechanical stretch on cardiomyocyte hypertrophic gene expression. Because cardiac hypertrophy can present itself as concentric or eccentric in vivo, we hypothesized that applying stretch in orthogonal directions to cardiomyocytes would produce divergent gene expression profiles. To investigate the directionally‐dependent cellular response to stretch, neonatal mouse cardiomyocytes were cultured on a deformable silicone membrane, which was micropatterned to create parallel grooves that aligned the cardiomyocytes. By adjusting the groove orientation relative to the stretcher axes, the primary direction of stretch could be applied parallel or transverse to the myofibril direction.

We conducted RNA sequencing (RNA‐Seq) to examine whole genomic gene expression changes after acute cardiomyocyte stretch. The results showed a more robust gene response to longitudinal than to transverse stretch. After 30 minutes of stretch, 349 and 1161 genes were considered differentially expressed (DE) from transverse and longitudinal stretch, respectively. After 4 hours, the number of DE genes increased to 962 and 3925. Gene ontology analysis indicated enrichment of cell cycle and cell growth by both directions of stretch; whereas only longitudinal stretch caused enrichment of actin cytoskeleton organization and lipid metabolism.

To further explore these gene expression changes, we constructed a logic‐based gene regulatory network from published data. From this network analysis, we identified SRF as a critical transcription factor in regulating longitudinal stretch‐induced gene changes. In addition, we performed Upstream Regulator Analysis in Ingenuity Pathway Analysis to identify putative transcription factors also responsible for stretch‐induced gene changes. These transcription factors include PPAR‐alpha, Klf15, and E2F1. The regulatory network model provides evidence that cardiomyocytes engage different transcriptional regulators in responses to different principal orientations of biaxial stretch.

Support or Funding Information

Supported by: NIH grants P50 GM094503, 1R01HL105242, GM103426, and HL103566 and CIRM grant RT3‐07899.
Progressive Thermal Preconditioning Efficiently Attenuates Rat Cardiac Ischemia/Reperfusion Injury
Authors: Chen Yueh‐Hsi
Published: November 2023

Progressive thermal preconditioning (PTP) to whole‐body hyperthermia is the protective effects on oxidative stress, which may have cardioprotective properties against ischemia/reperfusion (I/R) injury from efficiently diminish apoptosis and autophagy. We divided Male Wistar rats into non‐PTP control group, 72 h after a single or three consecutive cycles of PTP in a 42°C water bath (1–72, and 3–72 groups). Anesthetized male Wistar rats were subjected to 60 min of regional ischemia followed by 240 min of reperfusion. We measured cardiac O2−. amounts in vivo in response to left anterior descending coronary artery ligation for 1 h and reperfusion for 4 h. Cardiac function and injury was determined by microcirculation, electrocardiogram, infarct size and ventricular contractile function. PTP‐induced protective effects on oxidative stress, apoptosis‐and autophagy‐related mechanisms were examined by western blot and immunohistochemistry. Coronary arterial ischemia/reperfusion depressed cardiac microcirculation, induced S‐T segment elevation and increased infarct size in non‐PTP and PTP rats. Ischemia/reperfusion enhanced cardiac O2−. levels, erythrocyte and leukocytes infiltration, cytosolic cytochrome C release and the decreased mitochondrial Bcl‐2 expression. Cardiac injury increased Bax/Bcl‐2 ratio, cleaved caspase 3 expression and poly‐(ADP‐ribose)‐polymerase fragments leading to apoptosis formation, and promoted LC3‐II expression resulting in autophagy formation. PTP treatment elevated HSP70 and Bcl‐2 in the rat heart especially in the 3–72 group. PTP treatment significantly restored cardiac microcirculation and ventricular contractile function, and decreased inflammation, oxidative stress, apoptosis, autophagy and infarct size. PTP significantly reduces cardiac ischemia/reperfusion injury via the upregulating antioxidant, anti‐inflammatory, anti‐apoptotic and anti‐autophagic mechanisms.
Linking Metabolic and Structural Remodeling by Cardiac‐Specific Activation of the Mechanistic Target Of Rapamycin Complex 1
Authors: Davogustto Giovanni , Salazar Rebecca , Vasquez Hernan , Taegtmeyer Heinrich
Published: November 2023

Background

The heart remodels metabolically and structurally before it fails. Metabolically, the heart increases its reliance on carbohydrates for energy provision. Structurally, the heart hypertrophies to sustain increased hemodynamic stress. However, it remains unclear if both processes occur independently or if one is the consequence of the other. The activation of the mechanistic Target Of Rapamycin Complex 1 (mTORC1) pathway is closely tied to glucose uptake by the heart and also drives structural remodeling of heart muscle. With insulin stimulation or with increased workload, the glycolytic intermediate glucose 6‐phosphate (G6P) is required to activate mTORC1. Activation of this pathway leads to increased protein synthesis, hypertrophy and ER stress response leading to contractile dysfunction.

Objective

We examined whether sustained mTORC1 activation results in metabolic and structural remodeling in the heart, with special focus in the time course of these events.

Methods and Results

To do this we developed a Cre‐LoxP mouse model to achieve inducible, cardiac‐specific deficiency of the protein tuberin (TSC2), a member of the tuberous sclerosis complex, a key inhibitor of mTORC1. Echocardiograms were performed at 5, 15 and 20 weeks after induction. Mice were sacrificed 2, 4, 5 and 20 weeks after induction. Hearts were harvested, their weight recorded, rapidly freeze‐clamped and stored for further analysis. Genomic DNA, total RNA and proteins were extracted for qualitative PCR, real‐time PCR and immunoblotting respectively. G6P levels were measured by spectrophotometric enzymatic analysis using G6PDH coupled to NADPH production with extinction at 340 nm. Histologic analysis using H/E stain was used to determine left ventricle wall size and cardiomyocyte size. Immunoblotting of protein markers confirmed TSC2 knock out and activation of mTORC1 downstream targets two weeks after induction. G6P was increased at 2 and 4 weeks after induction, before any structural change could be detected. Serial echocardiograms showed evidence of concentric hypertrophy 15 weeks after induction. Histologic analysis using H/E confirmed concentric hypertrophic remodeling at 20 weeks.

Conclusion

The results indicate that inducible, sustained mTORC1 activation in the heart results in metabolic remodeling that precedes structural changes. We conclude that sustained mTORC1 activation is a key driver of the metabolic and structural remodeling preceding heart failure.

Support or Funding Information

Work in our laboratory is supported by grants from the U.S. Public Health Service (R01 2HL 61483‐10, R01 HL 073162). GD receives fellowship support from The University of Texas System Medical Foundation.
Body Mass Index is a Strong Predictor of Low Serum 25(OH)D Levels in Multiethnic Obese Children in Western Massachusetts
Authors: KANNAN SRIMATHI , Visintainer Paul , Ganguri Harish B , Wittcopp Chrystal
Published: November 2023

The objective of the study is to examine the association between low serum 25(OH)D levels (VDL) and BMI category in children, adjusting for sex, ethnicity, age, CVD risk markers, season and nutrient intakes. 99 obese (BMI> 95%) children aged 5–16 years participated in this cross sectional study conducted in a Pediatric Weight Management Clinic in Western Massachusetts. Nutrient intakes (Block Kids 2004 FFQ) and serum 25(OH)D were measured. Sunlight exposure, BMI, waist circumference (WC), BP, ethnicity, 25(OH)D, calcium, PTH, HbA1C, AST/ALT, fasting insulin, glucose, lipids and hsCRP data were obtained. Multivariable logistic regression was used to examine the relationship between VDL and BMI category, adjusting for potential confounders. Of 91 children completing the FFQ, mean 25(OH)D was 21.2 (±7.7) ng/mL and the mean BMI z‐score was 2.4 (±0.4). Using the Institute of Medicine (IOM) criteria, 25(OH)D was categorized into <15ng/mL (22.0%) and normal (≥15 ng/mL; 78%)., and BMIz, into >2.5 37.4%) and ≤ 2.5 (62.6%. Mean age 10.9±2.6 (SD) yrs, 22.0% African American, 26.4% Caucasian and 51.7% Hispanic, 52.8% female. Among those with BMI z‐scores less than 2.5, 15.8% had low 25(OH)D levels vs. 32.4% among those with BMI z‐scores at 2.5 or greater. That is, children with BMI z‐scores > 2.5 have 2.55 greater odds of having a low 25(OH)D level compared to those with BMI z‐scores <2.5 (OR=2.55, 95%CI: 0.93, 11.16; p=0.07). In logistic regression analysis, adjusting for gender, ethnicity, season, and age, children with BMI z‐scores >2.5 had a significant 5.14 greater odds of low 25(OH)D compared to children with BMI z‐scores ≤2.5 (95%CI: 1.30, 20.36; p = 0.02). Additional adjustment for energy‐adjusted nutrient values, waist circumference, and selected cardiovascular risk factors did not change this relationship. Obese children are at risk for vitamin D deficiency. This risk increases in children with severe obesity (BMIz > 2.5 SD) and is not significantly changed by season, ethnicity, nutrient intakes or sunlight exposure. More research is needed to further elucidate the mechanisms that influence vitamin D levels in this vulnerable population.

Support or Funding Information

This work was funded by UMASS‐Baystate Medical Center Collaborative Biomedical Research.
Urinary α‐CEHC Predicts α‐Tocopherol Status in Healthy Adults and Those with Metabolic Syndrome
Authors: Traber Maret G. , Leonard Scott W. , Mah Eunice , Bobe Gerd , Bruno Richard S.
Published: November 2023

More than 90% of Americans do not consume the estimated average requirement (12 mg) of vitamin E, but dietary intakes are poorly correlated plasma α‐tocopherol (α‐T) concentrations, which are apparently normal. Plasma α‐T is an insensitive biomarker of α‐T status because α‐T increases with lipid levels and in individuals with hyperlipidemia, circulating α‐T increases, but delivery of both lipids and α‐T to tissues is compromised. We hypothesized that α‐carboxyethyl hydroxychromanol (α‐CEHC) and α‐carboxymethylbutyl hydroxychroman (α‐CMBHC), physiologic metabolites of α‐T that are generated in a cytochrome P450‐dependent manner, would reflect α‐T status, and function as indicators of the lower α‐T bioavailability in adults with metabolic syndrome (MetS). Age‐ and gender‐matched healthy and MetS adults (n = 10/group) completed a randomized cross‐over pharmacokinetics study in which they co‐ingested 15 mg hexa‐deuterium labeled (d6)‐RRR‐α‐T with non‐fat milk, reduced‐fat milk, whole milk, or soy milk prior to measuring by LC‐MS the 24 h urinary α‐CEHC and α‐CMBHC excretion and 0–72 h plasma α‐T, α‐CEHC, and α‐CMBHC (ClinicalTrials.gov #NCT01787591). We showed previously that MetS adults had lower d6‐α‐T bioavailability (AUC0–72 h) and estimated d6‐α‐T absorption compared to healthy adults regardless of dairy milk co‐ingestion. We report herein that plasma d6‐α‐CEHC AUC0–72 h was 50% lower in MetS adults (P<0.05); 24 h urinary concentrations of unlabeled α‐CEHC and α‐CMHBC, and d6‐α‐CEHC and d6‐α‐CMHBC, were also ~50–60% lower in MetS compared to healthy adults (P<0.005). Plasma d6‐α‐T AUC0–72 h (μmol/L*h) was correlated with urinary excretion of d6‐α‐CEHC and d6‐α‐CMBHC (μmol/g creatinine, both P<0.0001), as well as with plasma d6‐α‐CEHC AUC0–72 h (nmol/L*h). In summary, labeled urinary metabolites of α‐T correlate highly with α‐T bioavailability of recently ingested α‐T. Urinary α‐CEHC, and α‐CMBHC may be useful biomarkers to non‐invasively assess α‐T adequacy, especially in populations with dyslipidemia‐associated impaired α‐T trafficking.

Support or Funding Information

Supported in part by DSM Nutritional Sciences, the National Dairy Council, and NIH (DK081761, UL1TR001070)
Extracellular Nucleotide Signaling Stimulates Vascular L‐Type Ca2+ Channel Activity And Vasoconstriction During Diabetic Hyperglycemia
Authors: Prada Maria Paz , Nystoriak Matthew A , Syed Arsalan , Nieves‐Cintron Madeline , Navedo Manuel F
Published: November 2023

Elevated extracellular glucose (i.e. hyperglycemia; HG) is a hallmark metabolic abnormality in diabetes and a major risk factor of vascular complications such as hypertension, stroke, and end organ damage. We recently discovered that HG amplifies intracellular Ca2+ concentration ([Ca2+]i) in vascular smooth muscle via PKA‐mediated potentiation of L‐type Ca2+ channels (LTCCs). Whereas elevation of [Ca2+]i by HG is prevented by ectonucleotidase activity, in this study, we tested the hypothesis that extracellular nucleotide signaling is required for PKA‐dependent potentiation of LTCC and vasoconstriction in response to HG. Using patch clamp electrophysiology and pressurized cerebral arterial diameter measurements, we found that HG‐potentiated LTCC current and endothelium‐independent vasoconstriction was associated with enhanced phosphorylation of CaV1.2 serine 1928 (S1928), a putative PKA target site in LTCCs. Consistent with our hypothesis, the increase in S1928 phosphorylation, vascular LTCC activity and vasoconstriction in the presence of HG was prevented by apyrase and a PKA inhibitory peptide, suggesting a mechanism involving the release of extracellular nucleotides and activation of PKA. Inhibition of the Gq‐coupled purinergic receptor P2Y6, which has been implicated in HG‐mediated increase in [Ca2+]i, failed to prevent the HG effects on S1928 phosphorylation, LTCC activity and vasoconstriction in response to HG. Altogether, our results indicate that extracellular nucleotide and PKA signaling is responsible for CaV1.2 S1928 phosphorylation, LTCC potentiation and vasoconstriction in HG. Considering our findings, we propose a potential functional association between PKA‐coupled purinergic receptors promoting LTCC activation and vasoconstriction in HG.

Support or Funding Information

This work was supported by grants NIH HL098200 and HL121059, and AHA‐14GRNT18730054 to MFN.
Enhanced Mobilization of Endothelial Ca2+ by SKA‐31 Contributes to the Restoration of Agonist‐mediated Vasorelaxation in Resistance Arteries from Type 2 Diabetic Rats
Authors: Kyle Barry D , Mishra Ramesh C , Braun Andrew P
Published: November 2023

Endothelial dysfunction is a common early pathogenic event in patients with type 2 diabetes (T2D). In isolated hearts from T2D Goto‐Kakizaki (GK) rats, we have observed that SKA‐31, a positive modulator of the endothelial Ca2+‐activated K+ (KCa) channels KCa2.3 and KCa3.1, increases total coronary flow and restores agonist‐evoked elevations in flow to the levels observed in Wistar control hearts. The goal of the current study was to investigate the cellular mechanisms by which SKA‐31 restores vascular responsiveness in T2D vessels. In cannulated, myogenically active cremaster and cerebral resistance arteries from 24 week old, male T2D GK rats, and small intra‐thoracic arteries from T2D patients, a threshold concentration of SKA‐31 (0.3 μM) enhanced the ability of 0.3 μM acetylcholine and 0.1 μM bradykinin to inhibit developed tone at 70 mmHg. This effect of SKA‐31 was not disrupted by treatment with L‐NAME (0.1 mM) and indomethacin (10 μM), demonstrating that endothelial nitric oxide synthase and cyclooxygenase activities do not contribute to SKA‐31 mediated enhancement of agonist‐evoked vasorelaxation in T2D arteries. Using single cell Fura‐2 fluorescence imaging, we observed that Ca2+ mobilization evoked by the muscarinic agonist carbachol (1 μM) was significantly impaired in acutely isolated endothelial cells from GK arteries compared with Wistar controls and that treatment with 0.3 μM SKA‐31 was able to reverse this defect in response. Ca2+ mobilization evoked by the SERCA inhibitor cyclopiazonic acid (20 μM) was not different in endothelial cells from Wistar and T2D GK arteries, suggesting that the internal Ca2+ store, per se, is not dysfunctional in GK endothelial cells. In summary, we have identified a novel cellular mechanism to restore agonist‐mediated inhibition of myogenic tone in T2D resistance arteries that involves “priming” of endothelial KCa channels by a small molecule activator (e.g. SKA‐31). Our data further reveal that SKA‐31 is able to augment agonist‐evoked Ca2+ mobilization in T2D endothelial cells, which likely contributes to the functional restoration of agonist‐evoked vasorelaxation in T2D arteries.

Support or Funding Information

This study was supported by research funding to APB from the CIHR.
Dynamic Regulation of Endothelial Cell‐Specific Molecule 1 in Diabetic Mouse Kidney
Authors: Zheng Xiaoyi , Soroush Fariborz , Hall Evan , Adishesha Puneeth , Bhattacharya Sanchita , Kiani Mohammad , Bhalla Vivek
Published: November 2023

Performing glomerular gene expression profiling in diabetic mouse models with differential susceptibility to nephropathy (DN), we identify endothelial cell‐specific molecule 1 (Esm‐1) as a novel candidate gene in the development of DN and sought to understand the functional role of Esm‐1 and how it may be regulated in diabetes. Esm‐1 is proposed to disrupt the interaction of lymphocyte function‐associated antigen 1 (LFA‐1) with intercellular adhesion molecule 1 (ICAM‐1) in vitro, suggesting Esm‐1 potentially blocks leukocyte infiltration, a known risk factor for DN. Therefore, we hypothesize Esm‐1 deficiency promotes leukocyte infiltration in susceptible mice and causes DN. Esm‐1 mRNA and protein expression profiles were tested in streptozotocin‐induced diabetic mice, and the functional relevance of Esm‐1 was tested in a bioinspired microfluidic assay (bMFA) which models leukocyte‐endothelial interactions ex vivo.

Results

In bMFA, pre‐treatment of leukocytes with Esm‐1 inhibits rolling and transmigration in a dose‐dependent manner. Consistent with the diminution of leukocyte infiltration in vitro, 4 weeks after induction of diabetes, Esm‐1 mRNA is markedly attenuated in glomeruli from DN‐susceptible, DBA/2J, compared to DN‐resistant, C57BL/6J, mice (relative fold: 0.085 ± 0.012, p<0.05). Secretion of glomerular Esm‐1 is also lower in DN‐susceptible mice (132.6 ± 41.6 vs 226.4 ± 79.3 pg/mL, p<0.05). These changes occurred in the background of a dynamic response of kidney Esm‐1 to diabetes. Urine Esm‐1 excretion increases in diabetic vs. non‐diabetic mice (307.7 ± 51.6 vs 102.3 ± 29.7 pg/mg creatinine, p<0.05), despite no increase in urine albumin and a ~50% decrease in circulating Esm‐1 in diabetic mice. Further in vitro studies in cultured glomeruli show that hyperglycemia significantly increases Esm‐1 mRNA and protein, but significantly less so in DN‐susceptible mice. In contrast to serum mouse Esm‐1, glomerular‐derived Esm‐1 is partially glycosylated, and the level of glycosylation is increased in DN‐susceptible vs. DN‐resistant mice. Glomerular conditioned media from DN‐susceptible vs. DN‐resistant mice also degrades Esm‐1 preferentially.

Conclusion

Esm‐1 is a potential blocker of leukocyte infiltration in glomeruli. High urine Esm‐1 level in diabetes is associated with diabetes/hyperglycemia stimulated glomerular Esm‐1 over‐production. But attenuated glomerular Esm‐1 over‐production is associated with high DN‐susceptibility. These data suggest that increased production of glomerular Esm‐1 would be sufficient to attenuate leukocyte infiltration and consequently, DN. Furthermore, we propose urine Esm‐1 as a non‐invasive candidate biomarker for resistance to DN.

Support or Funding Information

Larry L. Hillblom Foundation;

Holmgren Family;

NIDDK
Exercise Improves Microvascular Reactivity in the Comorbidity between the Metabolic Syndrome and Depression
Authors: Branyan Kayla , DeVallance Evan , Lemaster Kent , Chahal Ikttesh , Frisbee Jefferson , Chantler Paul
Published: November 2023

Metabolic syndrome (MetS) is increasing in prevalence at an alarming rate to where one out of every five Americans has multiple components of the disease. These components include dyslipidemia, hypertension, insulin resistance, and a chronic pro‐inflammatory state. Another disorder that is steadily rising is depression and it is estimated that 15.7 million adults in the United States suffer from its symptoms. Much is known about each disorder separately, but there is little evidence on comorbidity between the two. Both disorders are known to increase the risk of vascular dysfunction, and therefore also lead to an increase risk for poor cardiovascular outcomes. It is also known that exercise can be used as an intervention for MetS and depression. To determine if the comorbidity between MetS and depression have a confounding effect on microvasculature, specifically the gracilis arteriole, obese Zucker rats (OZRs) and lean Zucker rats (LZRs) were used as a model of MetS and healthy controls, respectively. An Unpredictable Chronic Mild Stress (UCMS) protocol was administered to mimic daily, chronic stress. OZRs and LZRs were then separated into experimental groups including: control, UCMS, exercise, and UCMS plus exercise. The gracilis arteriole was isolated and then hung in a pressurized vessel myobath to be exposed to increasing doses of acetylcholine, to assess endothelial function, and phenylephrine to examine adrenergic constrictor response. OZRs had a decreased overall gracilis reactivity from that of LZRs (p<0.01). The LZR‐UCMS group had decreased reactivity as compared to their controls (p<0.01) whereas OZR‐UCMS only had a slight decrease in gracilis reactivity. Exercise alone improved the gracilis reactivity in OZRs by 20% (p<0.01) but there was a significant increase in reactivity in the LZRs. In OZRs that were stressed and exercised, reactivity was recovered to control values. LZRs on the other hand had a 10% decrease in reactivity compared to their controls but exercise increased reactivity slightly when compared to the LZR‐UCMS group (p<0.01). TEMPOL only had an effect in the OZR groups which may suggest that adding a radical scavenger does not provide extra improvement in already healthy vasculature. These data suggest that the comorbidity between MetS and depression may have a confounding effect on healthy vasculature, but it does not necessarily increase microvascular dysfunction in already impaired vessels. It also suggests that exercise and radical scavenger intervention may be beneficial in improving microvascular reactivity in these disorders.

Support or Funding Information

AHA

NIH
Increased mTORC1 signaling in the vasculature of diet‐induced obese mice and obese humans
Authors: Reho John J , Guo Deng‐Fu , Wegman‐Points Lauren , Pierce Gary L , Rahmouni Kamal
Published: November 2023

Obesity‐induced hypertension is associated with vascular endothelial dysfunction. Recently our laboratory has implicated the mammalian target of rapamycin complex 1 (mTORC1) signaling in the regulation of the cardiovascular system. Specifically, activation of central mTORC1 triggered a significant increase in arterial pressure. Therefore, we tested the hypothesis that dysregulation of mTORC1 signaling is involved in the vascular endothelial dysfunction associated with obesity in mice and human patients. In diet induced obese mice (DIO), whole aortic homogenates demonstrated a significant increase in the mTORC1 signaling pathway compared to control as indicated by the increased phosphorylated levels of mTOR and its downstream targets S6‐kinase and S6 ribosomal protein (p<0.05) by Western blot. Immunohistochemical analysis, using a phospho‐S6 ribosomal protein antibody, of aortas from DIO mice revealed a substantial increase in mTORC1 signaling in the smooth muscle and the endothelium. Next, we isolated venous endothelial cells from normal‐weight (body mass index, BMI <25 kg/m2) and obese (BMI ≥30 kg/m2) humans and found increased levels of phosphorylated S6 ribosomal protein in obese compared with normal weight subjects (p<0.05), consistent with increased mTORC1 signaling. Stimulation of mTORC1 in cultured mouse aortas and resistance mesenteric arteries with the branched amino acid Leucine significantly increased mTORC1 signaling in both the endothelium and smooth muscle. Interestingly, cultured aortas stimulated with Leucine exhibited impairment in acetylcholine induced relaxation (Max. relaxation: 71.3 ± 2.1% vs 63.5 ± 1.9%; p<0.05), but no changes in relaxation responses evoked by sodium nitroprusside (Max. relaxation: 77.3 ± 2.3% vs 76.0 ± 4.5%) suggesting endothelial, but not smooth muscle, dysfunction. In order to selectively disrupt mTORC1 signaling in the vascular endothelium, we have generated endothelial‐specific mTORC1 knockout mice by breeding Tie‐2Cre mice with mice harboring floxed alleles of the unique regulatory associated protein of mTOR (Raptor) subunit (Tie‐2Cre/raptorf/f). Preliminary immunohistochemical analysis suggests endothelial specific knockout of mTORC1 signaling in these mice. Studies are ongoing to characterize the vascular function of these mice. We speculate that selective dysregulation of endothelial mTORC1 signaling is contributes to obesity‐induced endothelial dysfunction in both mice and humans.

Support or Funding Information

P01 HL084207
Comparison of Subjective Perception of Balance to Ocular Counter Roll in Veterans
Authors: Michalik Justyna , Migdal Kamila , Serrador Jorge , Fox Apollonia
Published: November 2023

Common symptoms of vestibular dysfunction are dizziness, nausea, feelings of vertigo as well as imbalance. Since these symptoms occur in many disorders, vestibular disorders are often under diagnosed in primary care settings (Grill, 2014). Vestibular dysfunction can lead to a greater difficulty in daily tasks and increase the risks of falls. While there are many risk factors for falls, loss of vestibular function greatly increases fall risk. We have previously assessed vestibular function in healthy adults from 21–97 by measuring ocular torsion (OT), a reflexive eye movement in response to body tilt (Serrador, 2009). Age was related to a significant reduction in OT. In addition, lower OT was strongly correlated with increased postural sway that has previously been found to predict fall risk. Previous literature has shown that veterans exposed to blasts and head injuries demonstrate vestibular symptoms, making veterans an ideal model to test for vestibular dysfunction. In the current study, we explored vestibular hypofunction by comparing ocular torsion (OT), an objective measure of otolith function, to a subjective perception of balance in veterans who have been exposed to blast and had head injuries as well as those who have not.

25 veterans (19 males; 6 females) from different conflicts volunteered for this study, ages 26–70 (mean age 52 ± 13.12). To measure ocularcounter roll, subjects were rotated ±25 deg in the roll plane while theirocular torsion was assessed using eye‐tracking goggles. For subjective perception of balance, participants were asked to complete several surveys about their balance and dizziness. The surveys consisted of an “Activities‐Specific Balance Confidence Scale”, “Dizziness Handicap Inventory” and “Vertigo SymptomScale”. 14 veterans were found to have low OT (below .14 degrees) while 11 were found to have normal ocular torsion. Out of the 14 veterans that were found to have low OT, 12 did not report having any balance issues or in conveniences during their day‐to‐day tasks across the various surveys used. A linear regression was run between the otolith function and questionnaires (R2 = .201, p=. 224). Interestingly, despite the lack of correlation, some questions, for example “riding an escalator without holding the rail”, showed a better correlation to the OT value, consistent with a task that would require otolithinputs. These data suggest that the self‐report questionnaires assessing balance function do not appear to provide a good assessment of otolith hypofunction. Thus future work is necessary to develop a questionnaire that would better assess otolithhypofunction.
Brain HMGB1 contributes to autonomic dysfunction
Authors: Masson Gustavo Santos , Nair Anand R , Dange Rahul , Soares Pedro Paulo Silva , Michelini Lisete Compagno , Francis Joseph
Published: November 2023

Pro‐inflammatory cytokines and activated microglia in the hypothalamic paraventricular nucleus (PVN) are involved in autonomic and cardiovascular control. Recently HMGB1 was identified as cytokine‐like peptide in the brain, which through the CXC receptor 4 (CXCR4) causes neuroinflammation and neurodegeneration. However, its role in autonomic dysfunction have been never investigated. We tested the hypothesis that intracerebroventricular (icv) administration of HMGB1 in normotensive rats causes microglia activation, autonomic dysfunction and cardiovascular abnormalities. Twelve‐week‐old male Sprague‐Dawley rats were implanted with a chronic lateral ventricle cannula and after 5‐days recovery, an arterial and venous catheters were inserted one day before recording. After baseline recordings in conscious freely‐moving rats, HMGB1 (0.38 and 3.8 μg icv) was injected and cardiovascular and autonomic parameters were continuously measured for 30 minutes. In another experimental group, HMGB1 was administered following pretreatment with icv AMD3100 (5 and 50 μg), a CXCR4 antagonist. Rats were euthanized; brains were quickly collected and PVN tissue harvested for western blotting and immunofluorescence assays. HMGB1 (3.8 μg icv) markedly increased baseline arterial pressure, heart rate, systolic arterial pressure variability and vasomotor sympathetic activity; these effects were accompanied by decreased vagal outflow and increased sympathetic activity to the heart, reduced baroreflex sensitivity and attenuated heart rate variability. In the PVN, administration of HMGB1 icv, rapidly activated microglia, increased the phosphorylation of p42/44 MAPK and IκB‐α and augmented TNF‐α and IL‐6 protein expression. All the hemodynamic function and PVN responses to HMGB1 were completely abolished by pretreatment with 50 μg AMD3100 given icv. These finding indicate that HMGB1 induces microglia–CXCR4–p42/p44MAPK–NF‐κB cascade, and contribute to the autonomic dysfunction.

Support or Funding Information

This work was supported by a LSU‐SVM Corp grant for Dr. Francis