bluuebunny/crossref_metadata_2025 huggingface dataset:
EHD2 Stabilizes the Surface Expression of Cardiac KATP Channels and Protects Against Ischemic Injury
Authors: Foster Monique , Jana Kundan , Bergman Lisen , Pennegård Elin , Ördberg Malin , Neubert Thomas , Coetzee William
Published: June 2021

Opening of ATP‐sensitive potassium (KATP) channels mediate ischemic preconditioning (IPC), which is an endogenous protection mechanism against ischemic injury. We found that internalization of KATP channels during ischemia is prevented by IPC. We investigated the role of EHD2 (identified in our proteomic assays as a KATP channel interacting protein) in KATP channel internalization. A dominant‐negative EHD2‐G65R caused Kir6.2/SUR2A KATP channels to redistribute in transfected COS‐7 cells. Both the dominant negative Rab11‐S25N and EHD2‐G65R led to accumulation of Kir6.2 in the endocytic recycling complex (ERC), suggesting that EHD2 controls exit from the ERC to the plasma membrane. Using biotinylation assays, we found that EHD2 decreased (whereas EHD2‐G65R accelerated) the rate of KATP channel endocytosis. Surface biotinylation experiments showed that EHD2 stabilized and EHD2‐G65R decreased the surface density of Kir6.2/SUR2A channels in HEK‐293 cells. Consistently, patch clamping demonstrated that EHD2 increases, whereas EHD2‐G65R decreases, KATP channel current density without affecting KATP channel properties. Similar data were obtained with isolated adult mouse cardiac myocytes infected with Ad‐mCherry‐EHD2 or Ad‐mCherry‐EHD2‐G65R. We employed a cellular model of pharmacological preconditioning, in which myocytes are pretreated with phenylephrine prior to ischemic pelleting. Adult mouse cardiomyocytes infected with Ad‐mCherry‐EHD2 resisted ischemic injury, whereas cell death was more prominent in Ad‐mCherry‐EHD2‐G65R. Our data demonstrate that EHD2 positively regulates KATP channel surface expression and participates in the protection afforded by preconditioning.
Absence of Gravin Scaffolding to the β2‐AR Protects against Heart Failure and its Protective Effects are Additive to Carvedilol Treatment Alone
Authors: Singh Sonal , Suryavanshi Santosh , Diaz Diaz Andrea , McConnell Bradley
Published: June 2021

Gravin, an A‐Kinase Anchoring Protein (AKAP) is a scaffolding protein in the heart involved in the modulation of cardiac contractility. Specifically, gravin scaffolds PKA, PKC, β‐arrestin and various other proteins to β2‐adrenergic receptors (β2‐ARs) and functions to modulate contractility by mediating β2‐ARs desensitization. We previously found that disruption of gravin scaffolding, using gravin knockout (gravin‐t/t) mice, reduced cardiac hypertrophy induced by chronic β‐AR stimulation with isoproterenol (ISO; 60mg/kg/day for 14 days). However, for disruption of gravin scaffolding to be considered a therapeutic target, it will be important to show that inhibiting gravin scaffolding is a superior approach over conventional therapies. Hence, the goal of this study was to study the effects of carvedilol (CARV) in wild‐type (WT) versus gravin‐t/t animals; where CARV is the gold standard β‐blocker used to treat human heart failure (HF). WT and gravin‐t/t mice were treated with ISO chronically for 2‐weeks to induce heart failure and then treated with CARV for additional 3‐weeks. We found that heart‐to‐body weight (HW/BW) ratio, an indicator of cardiac hypertrophy, was significantly lower in the gravin‐t/t‐ISO versus WT‐ISO mice. Following CARV treatment, HW/BW was further decreased in the gravin‐t/t‐CARV mice. In addition, hemodynamic pressure‐volume measurements of contractility (dp/dtmax, Ees, cardiac output) and relaxation (Tau) were significantly greater in gravin‐t/t CARV mice as compared to WT‐CARV mice. Our data suggests that the absence of gravin scaffolding in gravin‐t/t mice has a protective role in HF, exceeding the positive effects of CARV alone.
Biventricular Differences in β‐adrenergic Receptor Signaling Following Burn Injury
Authors: Guillory Ashley , El Ayadi Amina , Mifflin Randy , Herndon David , Finnerty Celeste
Published: June 2021

Burn injury has been shown to have detrimental effects on the myocardium primarily due to over‐activation of β‐adrenergic receptors (β‐AR). Autopsy reports from our institution reveal that patients often suffer from right ventricle (RV) failure. Since burn injury affects β‐AR signaling in the left ventricle (LV), we proposed that β‐AR signaling may also be altered in the RV.

We used a rat model of 60% total body surface area burn. Ventricles were isolated at 7 days post‐burn. We examined the expression of β‐ARs and downstream proteins via Western blotting. cAMP production and PKA activity were measured in membrane and cytosolic fractions, respectively, using enzyme immunoassay kits.

β1‐AR expression was significantly increased in the RV following burn injury compared to non‐burned RV but remained normal in the LV (p=0.0002). Similarly, Gαs expression was increased in the RV post‐burn but not in the LV (p=0.04). There were no differences in β2‐AR expression among the groups. Gαi expression was significantly higher in the LV post‐burn compared to non‐burned LV, burned RV and non‐burned RV (p<0.0001). Similarly, cAMP production and PKA activity were significantly lower in the LV post‐burn compared to non‐burned LV, burned RV and non‐burned RV (p=0.0063, 0.0042, respectively).

These data indicate that burn injury affects the β‐AR signaling pathway in the RV independently of the LV. Additionally, non‐canonical β‐AR signaling may be involved as cAMP production and PKA activity were unchanged.

This study was supported by grants from the National Institutes of Health (P50‐GM60338, KL2RR029875, UL1RR029876) and Shriners Hospital for Children (80100, 84291, 80500, 84202, 71001).
TUG Mediates GLUT4 Translocation by AMP‐Activated Protein Kinase in the Heart
Authors: Ma Yina , Wang Jinli , Wang Yanqing , Bogan Jonathan , Li Ji
Published: June 2021

Introduction

Ischemic heart disease, is mainly caused by reduced nutrient availability. Increasing nutrient uptake becomes a key approach to improve cardiomyocyte survival during the ischemia and reperfusion (I/R) period. TUG (tether containing a UBX domain, for GLUT4, 60 KDa) is a regulator of GLUT4 trafficking, which is cleaved to mobilize GLUT4 from intracellular compartments to the cell surface. The energy sensor AMP‐activated protein kinase (AMPK) is known to play an important cardioprotective role during myocardial I/R by regulating GLUT4 translocation. Antithrombin (AT) exhibits a cardioprotective function severed as AMPK activator.

Hypothesis

TUG is one of the downstream targets of AMPK, which can be phosphorylated by hypoxia/ischemia‐induced as well as AT‐induced AMPK activation.

Methods

In vitro hypoxia chamber and in vivo I/R models were used to analyze AMPK/TUG interactions. Ex vivo isolated mouse heart perfusion Langendorff system was used to measure glucose metabolism.

Results

In HL‐1 cardiomyocytes, TUG proteins were phosphorylated by activated AMPK during hypoxia. Moreover, TUG siRNA knockdown the TUG of HL‐1 cells caused significantly increased cell surface GLUT4 and glucose uptake. The ex vivo heart perfusion data demonstrated that AT triggered AMPK activation and significantly increase glucose uptake and GLUT4 translocation during I/R. Moreover, AT treatment increased abundance of a TUG cleavage product (42KDa) in response to I/R. All of these glucose transporter events are blunted in the AMPK kinase dead transgenic hearts.

Conclusions

Cardiac AMPK activation via AT during the I/R period stimulates TUG cleavage and causes the dissociation between TUG and GLUT4, followed by the enhanced glucose uptake.
Systems pharmacology guided platform development for the integrated Microphysiological Systems
Authors: Yu Jiajie , Cirit Murat , Carrier Rebecca , Chen Kelly , Cilfone Nicholas , Coppeta Jonathan , Griffith Linda , Hughes David , Large Emma , Lauffenburger Douglas , Lever Amanda , Mescher Mark , Prantil‐Baun Rachelle , Sarkar Ujjal , Stokes Cynthia , Tannenbaum Steven , Wishnok John
Published: June 2021

The current drug clinical development is suffering a high attrition rate. More robust preclinical tools are needed to identify unpredicted toxicity and efficacy problems in the early stage of the drug development. Microphysiological Systems (MPS) program aims to develop in vitro interactome system to mimic physiological responses of drugs and provide more reliable preclinical results that is predictive of clinical outcomes. Systems pharmacology approach was used to analyze experimental data and guide integrated MPS platform development. Experimental results from MPSs were analyzed to obtain mechanism‐based information that could not be easily interpreted without systems pharmacology models. A series of data‐driven computational models for 1‐, 2‐, and 4‐MPSs was also developed to study platform operations under physiological conditions. Moreover, our systems pharmacology framework also assisted researchers to develop integrated MPS platform in physiological manner. Overall, systems pharmacology has been accepted as a very useful approach for quantitatively analyzing experimental results, experimental design and accelerating physiological Microphysiological Systems platform development.
Revisiting the Promethean Dream: The Role of Activation‐induced Cytidine Deaminase in the Induction to Pluripotency
Authors: Chakraborty Ritayan , Chaudhuri Jayanta
Published: June 2021

Upon encountering antigens in the germinal centers of peripheral lymphoid organs, mature B‐lymphocytes undergo class switch recombination (CSR). CSR alters B cell expression between different immunoglobulin types. This mechanism requires activation induced cytidine deaminase (AID). However, recent speculation into AID activity has led to the conclusion that AID can function as an indirect demethylase.

The aim of the experiment is the elucidation of AID's molecular mechanism during the development of induced pluripotent stem cells (iPSCs). We hypothesize that when a cocktail of 4 transcription factors Oct4, Klf4, Sox2 and cMyc are introduced into fully differentiated cells using a retroviral vector, the cell undergoes a reprogramming process that promotes its conversion to an iPSC. AID is thought to mediate this conversion. Using recombinant mice for AID mutations and chemically transfecting the retroviral cassette with Polybrene, AID expression was tested via Flow Cytometry and Western Blotting. Results with an LSRFortessa X‐20 Cell Analyzer and Zeiss Confocal lens microscope at 150x. The results indicated that upon infection with OKSM factors only AIDpositive cells began reprogramming showing certain early pluripotent factors and a down‐regulation of CSR while AID knockout cells failed to maintain the pluripotent state for an extended period of time.
Mechanosignal Transduction via NOX2 with Lung Ischemia Reperfusion: Lessons for Lung Transplant
Authors: Tao Jian‐Qin , Sorokina Elena , Feinstein Sheldon , Cantu Edward , Christie Jason , Fisher Aron , Chatterjee Shampa
Published: June 2021

I/R injury, a major cause of lung transplant failure, occurs predominantly due to recruitment of polymorphonuclear neutrophils (PMN) into the (donor) lung endothelium from the recipient's circulation post‐transplant surgery. Endothelial signaling is pivotal in driving the recruitment and adherence of PMN to the vascular wall. We used our past research, expertise and knowledge on pulmonary endothelial mechanosignaling (wherein we unraveled the cascade associated with ischemia to show a KATP closure induced endothelial NADPH oxidase 2 or NOX2 activation and reactive oxygen species, ROS, production) to evaluate how stop and restart of flow lead to PMN adherence. We show here (using in vitro, in situ and in vivo models of lung I/R) that endothelial NOX2 activation (as monitored by ROS production in vitro, in situ and oxidative damage in vivo) is linked to expression of adhesion molecules (CAMs) and to the recruitment of PMN. The mechanosignal can be blocked by preventing NOX2 activation either directly by blocking NOX2 assembly (with apocynin or with MJ33 that blocks the PLA2 activity of peroxiredoxin 6) or by blocking the upstream events (KATP channel); this has potential application in lung transplantation.
Greater Yield of Subsarcolemmal Mitochondria from Skeletal Muscle of Obese Subjects with No Change in Intrinsic Mitochondrial Function
Authors: Kras Katon , Willis Wayne , Tran Lee , De Filippis Elena , Roust Lori , Katsanos Christos
Published: June 2021

Whether obesity influences content and functional capacity of skeletal muscle mitochondria (MITO) remains unresolved. In this study, biopsies of vastus lateralis from lean and obese human subjects were used to isolate subsarcolemmal (SS) and intermyofibrillar (IMF) MITO. IMF MITO were liberated using a protease. The SS and IMF were pelleted separately, using differential centrifugation. Skeletal muscle from obese subjects gave higher MITO protein yield, per g wet muscle vs. skeletal muscle from lean subjects in the SS but not in the IMF. There was a trend for higher isolated yields of citrate synthase (CS) activity, a marker for MITO content, in the muscle of obese subjects vs. lean subjects in SS, but not in IMF MITO. O2 consumption (JO) and ATP production rates measured with NAD‐linked substrates (malate+pyruvate+glutamate), were not different in obese vs. lean in either SS or IMF MITO expressed either per mg MITO protein or per unit CS activity. The similarities in MITO function observed in SS and IMF from both lean and obese human muscle, suggests comparable functional capacity per unit MITO, regardless of the location within the myocyte, or whether the MITO reside in skeletal muscle from lean or obese subjects. However, higher total yield of both protein and CS activities in the SS MITO from the muscle of obese subjects, point to the possibility for greater content of SS MITO in muscle of obese individuals. This conclusion must be qualified; these results may be due to a greater fractional yield of MITO during the isolation procedure of skeletal muscle from obese subjects.
MiR‐133a Mitigates Mitophagy in Ins2+/‐ Diabetic Heart
Authors: Nandi Shyam Sundar , Mishra Paras Kumar
Published: June 2021

To test the hypothesis that inhibition of miR‐133a induces mitophagy in diabetic hearts and it is ameliorated by overexpression of miR‐133a, we used four in vivo groups: C57BL/6J, Akita (T1D), Akita+miR‐133a, and Akita+scramble; and four in vitro groups of HL1 cardiomyocytes: low (LG‐5mM), high (HG‐25mM) doses of D‐glucose, HG+miR‐133a, and HG+scramble; and determined mitoSOX generation, depolarization of damaged mitochondria (ΔΨ, by JC‐1), mitochondrial fission (by DRP1 and Parkin), autophagosome formation (by Beclin1, ATG14, ATG3, LC3B) and its lysosomal degradation (by RFP‐GFP‐LC3B) using qPCR, Western blotting, immunofluorescence, and transmission electron microscopy (TEM). Our results demonstrate that in HG cardiomyocytes, mitoSOX generation, depolarization of mt‐ΔΨ and DRP1 protein are elevated by 2.5±0.3, 1.44±0.08 and 1.94±0.29 folds, respectively, but ameliorated (mitoSOX:4.9±0.04, mt‐ΔΨ:2.11±0.08, and DRP1:0.85±0.17 fold down regulation) by overexpression of miR‐133a. The levels of Beclin1, ATG14, ATG3 and LC3B are increased in HG by 1.21±0.09, 1.46±0.24, 1.63±0.2, and 1.44±0.02 folds but mitigated by 1.47±0.08, 1.16±0.03, and 2.19±0.2, 1.54±0.02 folds respectively. The lysosomal degradation of autophagosome is elevated in HG by 4.51±0.9 fold but down regulated by 2.07±0.2 fold in miR‐133a overexpressing cardiomyocytes. DRP1 and Parkin proteins are up regulated by 1.94±0.01 and 1.99±0.37 folds in Akita but mitigated 2.5±0.02 and 2.25±0.43 folds by miR‐133a, respectively. TEM elicited increased mitophagy in Akita but restored in Akita+miR‐133a. We conclude that attenuation of miR‐133a increases mitoSOX, depolarizes mt‐ΔΨ and induces mitophagy, whereas overexpression of miR‐133a restores their levels and ameliorates mitophagy in diabetic hearts.
Differential responses in brachial and ankle blood pressure during blood pressure reactivity tests
Authors: Pearman Miriam , Lim Ji Sok , Park Wonil , Alkatan Mohammed , Machin Daniel , Tanaka Hirofumi
Published: June 2021

Blood pressure (BP) reactivity has been assessed by a variety of stimuli, including physical, psychological, and physiological perturbations, that can be further classified into cardiac or vascular in regard to the effector organs. In many BP reactivity studies, changes in brachial BP are taken as systemic changes and are combined with leg measurements to assess vasoactive states. Currently, it is unknown if leg BP behaves similarly to arm BP.

Purpose

We compared BP changes between the brachial and post‐tibial arteries during acute BP perturbations.

Methods

Thirty apparently healthy subjects (57% males, 46±4 years) were studied. Both brachial and ankle systolic BP (SBP) were measured simultaneously with the oscillometric technique during resting baseline, head‐up tilt (HUT), head‐down tilt (HDT), mental stress (MS), isometric hand‐grip exercise (HG), and a cold pressor test (CPT).

Results

Brachial (bSBP) and ankle (aSBP) SBP at rest were significantly and highly correlated (r=0.81). Changes in bSBP were significantly and positively associated with changes in aSBP in response to HUT (r=0.46), HDT (r=0.53), MS (r=0.70), HG (r=0.63), and CPT (r=0.75). The magnitudes of bSBP and aSBP changes were not different n response to MS, HG, and CPT since all were within 5 mmHG. There were significant differences in the magnitude of changes in bSBP and aSBP in response to HDT (12±1 mmHg) and HUT (40±2 mmHg).

Conclusion

Both brachial and ankle BP respond similarly in many vascular reactivity maneuvers. During head‐down and head‐up tilt, however, differences in their responses may be quite large, presumably due to the hydrostatic pressure gradients created during the maneuvers.

Supported by Fukuda Denshi.
Loss of CD47 Attenuates Angiotensin II Mediated Hypertension
Authors: Mutchler Stephanie , Isenberg Jeffery , Straub Adam
Published: June 2021

CD47 plays a role in vascular reactivity by inhibiting both nitric oxide (NO) production and signaling. Because altered NO signaling is implicated in cardiovascular disease and hypertension, we explored the role of CD47 in the development of angiotensin II (AngII) mediated hypertension. In anesthetized CD47‐/‐ mice, AngII injection failed to elicit the increase in blood pressure seen in a WT mouse. Expression of the AT1R receptor was measured in homogenized vessels via Western blotting, and it was determined that levels were not significantly different between WT and knockout animals, suggesting decreased response was not due to lack of receptor availability. To further characterize this lack of response, isolated TDAs from both WT and CD47‐/‐ mice were cannulated and pressurized. Vessel diameter was assessed in response to increasing doses of AngII. Unexpectedly, we found that vessels from CD47‐/‐ mice displayed decreased sensitivity to AngII‐mediated vasoconstriction compared to WT vessels. Constriction to KCl was similar in vessels from WT and CD47‐/‐ animals, demonstrating that the contractile mechanism was still fully intact in the knockout animal. Taken together, this data suggests CD47 is important for AngII‐mediated vasoconstriction and therefore has the ability to effect systemic tone and blood pressure. We are actively investigating the molecular mechanisms governing this relationship, and plan to explore blood pressure in conscious animals via radiotelemetry units as well as whether germ line CD47 deletion and therapeutic blockade of CD47 protects against AngII‐induced organ remodeling in the heart and kidneys.

Source: NIH R00 HL‐11290402, NIH R01 HL‐108954
NF‐қB Activation is Required for Thyroid Hormone Induced Cardiomyocyte Hypertrophy
Authors: Takano Ana Paula , Moriscot Anselmo , Barreto‐Chaves Maria Luiza
Published: June 2021

The nuclear factor NF‐қB promotes inflammatory response and mediates cardiac growth by different hypertrophic stimuli. We have previous results obtained by microarray experiments indicating that thyroid hormone (triiodotyronine, T3) up‐regulates pro‐inflammatory genes. Considering that, we investigated the influence of T3 on NF‐қB signaling and the possible participation of this nuclear factor in cardiomyocyte hypertrophy induced by T3. Primary cultures of neonatal rat ventricular cardiomyocytes were treated with T3 for 10, 30 minutes and 24 hours and the NF‐қB signaling was analyzed by western blot. Also, cardiomyocytes were treated with an inhibitor of NF‐қB (JSH‐23) followed by treatment with T3 for evaluation of mRNA expression of atrial natriuretic factor (ANF) and cell surface area analysis. Data were presented as mean ± SD and were analyzed using one‐way ANOVA and Tukey multiple comparisons test. Values of p<0.05 were considered statistically significant. T3 rapidly increased the nuclear protein expression of NF‐қB without alterations on IKK and IқB expression. The inhibition of NF‐қB reduced both ANF mRNA levels and the surface area of cardiomyocytes compared to T3 treated cells. These results confirm that NF‐қB is required for development of cardiomyocyte hypertrophy by T3. The possible interactions of NF‐қB with genes associated with cardiac growth are being investigated. Financial support: Fapesp (2013/22480‐4 and 2011/23352‐4).
Effects of Epicatechin on AMPK, GLUT4 and TNF‐α in the onset of Type 2 Diabetes
Authors: Gonzalez Nelson , Go Yamazaki Katrina
Published: June 2021

An abnormally high level of blood glucose is the key characteristic of type 2 diabetes (T2D). Several studies have established that this results from decreased translocation of glucose transporters. Due to the consequences of T2D, i.e. cardiovascular disease, the glucose transporter type 4 (GLUT4), found in both skeletal and cardiac tissue, is of important interest. T2D has been linked to energy deficiency leading to cardiovascular disease, possibly due to the lack of the metabolic substrate glucose. A key protein in an insulin independent pathway is 5′ adenosine monophosphate‐activated protein kinase (AMPK), which is directly involved with the translocation of GLUT4 and energy deficiency. Additionally, literature has suggested that the pro‐inflammatory cytokine tumor necrosis factor‐α (TNF‐α) may be associated in the development of insulin resistance, by targeting insulin signaling markers, and heart inflammation, decreasing overall cardiac functionality. Using an in vivo high fat, streptozotocin (STZ) induced T2D model, we investigated whether (‐)‐epicatechin (EPI), a naturally occurring flavonoid with demonstrated anti‐diabetic properties, altered the relative expression of the aforementioned proteins in heart tissue samples between sham and diabetic H20 or EPI treated animals. Utilizing western blot assays, we observed a reduced expression of GLUT4 and TNF‐α and an increased expression in AMPK in T2D models treated with EPI in comparison to T2D models treated with H2O. Our results suggest that EPI is playing a role in the increased expression of AMPK and decreased expression of GLUT4 and TNF‐α in the on set of T2D.
ASK1 deficiency attenuates hyperoxia‐induced inflammation and cell apoptosis in the lung
Authors: Fukumoto Jutaro , Hosseinian Nima , Priyanshi Patel , Cho Young , R. Cox Ruan , Lakshimi Galam , Soundararajan Ramani , F. Lockey Richard , Kolliputi Narasaiah
Published: June 2021

Rationale

Apoptosis signal‐regulating kinase 1 (ASK1), a member of the MAPK kinase (MAP3K) family, is activated by various stimuli including oxidative stress, ER stress, and DNA damage. Inspiration of high oxygen fraction is a palliative therapy to counteract acute lung injury (ALI). Our previous data indicates that ASK1 plays a critical role in this process. However, it is unclear whether or not deletion of ASK1 in vivo protects against HALI. We investigated whether ASK1 deletion would lead to protection against HALI.

Methods

ASK1 KO mice and control littermates were exposed to 100% oxygen for 48‐72 hours and euthanized to collect bronchoalveolar lavage (BAL) fluid for cell counts and cytokine measurements, and lung tissue collected for assessment of lung epithelial cell apoptosis.

Results

BAL fluid analysis revealed a significant decrease in IL‐1β level in KO mice. Differential cell count shows suppressed inflammatory cell infiltration in KO mice. Lung epithelial cell apoptosis was attenuated in ASK1 KO mice compared to WT mice as determined by TUNEL staining. Moreover, ASK1 KO mice exhibited significant increase in protein concentration in BAL fluid.

Conclusion

ASK1 has a proinflammatory profile. Meanwhile, the permeability increase was observed in ASK1 KO mice which indicate that hyperoxia‐induced increase in permeability is independent of ASK1. Our data suggest that ASK1 can be a novel molecular target for ALI treatment.

Funding source: This work was funded by the American Heart Association National Scientist Development Grant 09SDG2260957 and National Institutes of Health R01 HL105932 and the Joy McCann Culverhouse Endowment to the Division of Allergy and Immunology.
Inhibition of MiR‐92 Mimics Arterial Aging
Authors: Hazra Sugata , Henson Grant , Reihl Kelly , Bramwell R , Donato Anthony , Lesniewski Lisa
Published: June 2021

MicroRNAs (miRs) are small non coding RNAs that are important regulators of aging and cardiovascular diseases. One miR, miR‐92, is important in developmental vascular growth and tumor genesis and has putative targets, i.e. tumor necrosis factor alpha receptor (TNFR)‐1 and collagen type 1 that are involved in arterial dysfunction. We hypothesized that reduced expression of miR‐92 contributes to age related arterial dysfunction characterized by endothelial dysfunction and large artery stiffness. miR‐92 is reduced ~40% (p<0.05, RT‐PCR) in aortas of old (29 mo, n=13) compared to young (6 mo, n=11) B6D2F1 mice, an established model of vascular aging. To determine if reduced miR‐92 could contribute to arterial dysfunction; miR‐92 was inhibited in vivo in young mice using antagomirs (I.P., 4‐6 weeks). Antagomir treatment reduced aortic miR‐92 expression by 33% (p<0.01) and was associated with a concomitant 48% increase in TNFR1 (Western blot, p<0.05) and a 19% increase in type1 collagen (p<0.01). miR‐92 inhibition also increased arterial stiffness (309±13 vs. 484±52 cm/s, p<0.05, pulse wave velocity) and reduced endothelial dependent dilation (max: 92±1 vs. 76±5 %, p<0.01) in response to acetylcholine (ACh). Treatment with the nitric oxide (NO) synthase inhibitor L‐NAME revealed that the reduction in ACh dilation with antagomir was due to reduced NO bioavailability. These results suggest that experimental reductions in arterial miR‐92 mimic the arterial aging phenotype. In conclusion we speculate that modulating miR‐92 may provide a therapeutic strategy to improve age‐related arterial dysfunction.
Toll‐like receptor 9 Activation Contributes to Decreased Autophagy in Hypertension
Authors: McCarthy Cameron , Wenceslau Camilla , Goulopoulou Styliani , Webb R Clinton
Published: June 2021

Autophagy is a catabolic process for cell survival in times of stress. However, dysregulation for this process can have detrimental effects on cell survival. In fact, insufficient autophagy has been proposed as a mechanism of apoptosis due to the accumulation of proteins and defective organelles. We have observed that spontaneously hypertensive rats (SHR) have elevated circulating levels of the damage‐associated molecular pattern mitochondrial DNA (mtDNA), diminished activity for the enzymes responsible for its degradation, deoxyribonuclease (DNase) I and II, and these novel phenotypes contribute to the activation of Toll‐like receptor (TLR)9. Therefore, we hypothesized that expression of representative proteins associated with autophagy are decreased in SHR and that TLR9 activation contributes to this down regulation. We observed decreased expression of autophagic proteins in the aorta of SHR (Beclin‐1, ATG5, and LC3‐I and ‐II) (p<0.05 vs. WKY). Moreover, in vivo treatment of normotensive rats with a synthetic TLR9 agonist (ODN2395; 0.1 μg/i.p.), increased systolic blood pressure (mmHg, Veh: 141±3 vs. ODN2395: 154±3, p<0.05) and decreased the expression of ubiquitin‐like protein and autophagosomal marker LC3‐II in both resistance arteries and the aorta (p<0.05 vs. Vehicle). These results suggest that stimulation of TLR9 may be a contributing mechanism for the reduction in the autophagic machinery observed in SHR. Therefore, targeted inhibition of mtDNA and/or TLR9 could prevent the dysregulation of autophagy in hypertension.
Anti‐aging Gene Klotho Deficiency Causes Arterial Calcification via Activating the Bone Morphogenetic Protein Signaling
Authors: Lin Yi , Sun Zhongjie
Published: June 2021

Background

Arterial calcification is an important risk factor for arterial stiffening, hypertension, stroke, and coronary heart disease. Klotho‐deficient mice display severe arterial calcification but the mechanism remains to be determined.

Methods & Results

We found that Klotho‐deficient mice developed elastin fiber fragmentation and smooth muscle cell (SMC) apoptosis and arterial calcification. Aortas of Klotho (‐/‐) mice demonstrate higher levels of bone morphogenetic proteins (BMP2 and BMP4) and runt‐related transcription factor 2 (RUNX2, a key transcription factor for bone formation). Given that Klotho deficiency‐induced hyperphosphotemia may cause arterial calcification, Klotho deficient mice were given low phosphate diet (0.2%). Interestingly, Low phosphate diet normalized blood phosphate levels and abolished calcification in lungs and kidneys, but it did not prevent the development of aortic calcification in Klotho‐deficient mice. In cultured mouse aortic SMCs, KL‐deficient serum promoted BMP2‐induced calcification, protein expressions of Pit2 (phosphate transporter) and Runx2, and phosphorylation of Smad1/5/8 and Smad2/3. Interestingly, treatment with KL abolished the BMP2‐induced morphogenetic effects.

Conclusion

Klotho deficiency‐induced arterial calcification is an active process which requires activation of the BMP2 signaling pathway.
Hyperhomocysteinemia (HHcy) Causes Mitochondrial Dysfunction and Epigenetic Modifications Leading to Skeletal Muscle Weakness
Authors: Veeranki Sudhakar , Winchester Lee , Tyagi Suresh
Published: June 2021

HHcy has been implicated in reduced tissue perfusion and elderly frailty, but the underlying mechanisms are poorly understood. We recently demonstrated that HHcy attenuates ischemic angiogenic responses in muscles and delays vasculogenesis leading to muscle atrophy. Lack of prompt Akt activation led to dysregulation of VEGF, HIF‐1α, and PGC‐1α levels in ischemic muscles and unleashed FOXO transcription. Now, using HHcy mice and C2C12 cells, we investigated intrinsic mechanisms behind HHcy induced skeletal muscle weakness and fatigability. An exercise regimen was employed to reverse HHcy induced changes. The HHcy mice exhibited more fatigability, and generated less contraction force. No significant changes in muscle morphology were observed. However, there is a corresponding reduction in large muscle fiber number in HHcy mice. Excess fatigability was not due to changes in key enzymes involved in metabolism, but was due to reduced ATP levels. A marginal reduction in dystrophin levels along with a decrease in mtTFA was observed. There was also an increase in the mir‐31, and mir‐494 quantities that were implicated in dystrophin and mtTFA regulation respectively. Amount of NRF‐1, transcriptional regulator of mtTFA, was also significantly decreased. Exercise reversed most of the changes. There was also enhancement in mir‐494 levels and a concomitant decline in mtTFA protein quantity after homocysteine treatment along an increase in DNMT3a & 3b proteins and global DNA methylation levels. Interestingly, miR‐494 inhibition was also implicated in neovascularization. The results suggest that HHcy plays a causal role in enhanced fatigability and delayed ischemic muscle responses through mitochondrial dysfunction and epigenetic changes.
Nitroglycerin‐induced tolerance in rat conduit artery and vein
Authors: Paulo Michele , Daruge Grando Marcella , Vercesi Juliana , Bendhack Lusiane
Published: June 2021

Clinical use of nitroglycerin (GTN) is limited because its long‐term therapy results in the development of nitrate tolerance. The etiology of nitrate tolerance is controversial and poorly understood. This study aimed to investigate the mechanisms of GTN‐induced tolerance and to compare them in aorta and vena cava from GTN tolerant rats. In vivo tolerance for GTN was induced by 3 days treatment with GTN (150 mg/kg/day). We evaluated the maximum relaxing effect (ME) and the potency (pD2) induced by GNT in aorta and vena cava from tolerant and control rats. The endothelial NOS3 and soluble guanylyl‐cyclase (sGC) protein expression was accessed by Western Blot. The procedures are according to the Ethics Committee. Differences were considered significant for P<0.05. GTN treatment reduced the pD2 of GTN from 14.3±0.8, n=6 to 12.8±0.6, n=6, whereas ME was not different between control and tolerant rat aorta. In vena cava, ME was lower in tolerant rats (35.2 ± 2.5%, n= 6, P<0.05) as compared to control rats (73.1 ± 5.7%, n=6). sGC expression was similar in aorta and vein from control and tolerant rats. However, NOS3 expression was higher in aortas from tolerant (1.08 ± 0.06 n= 6, P<0.05) than in control animals (0.66 ± 0.04, n=7). Similarly, NOS3 expression was higher in veins from tolerant (1.08 ± 0.06 n= 6, P<0.05) than in control animals (0.66 ± 0, 04, n= 7). Therefore, GTN‐induced tolerance in rat aorta and vein involves NOS3 but not sGC.
HDAC Expression and Activity in Vascular Smooth Muscle Cells is Regulated by Glucose‐6‐Phosphate Dehydrogenase
Authors: Dhagia Vidhi , Gupte Rakhee , Gupte Sachin
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD), a key enzyme in the glucose metabolism, regulates vascular smooth muscle cell (VSMC) phenotype and function. In proteomic analysis, we discovered that G6PD formed a complex with histone acyltransferase (KAT6A) and deacetylase (HDAC5) in VSMCs. Therefore, we investigated whether HDAC expression and activity is regulated by G6PD in pulmonary artery and aortic smooth muscle cells. G6PD knock down decreased HDAC5 expression and G6PD inhibition decreased HDAC activity by 70‐80%. G6PD inhibition also decreased cell proliferation and increased myocardin and myocardin‐dependent gene expression. In summary, we propose that: 1) G6PD‐dependent HDAC function is involved in the regulation of VSMC‐specific genes and VSMC phenotype, and 2) G6PD‐dependent HDAC activation could be important in down‐regulating myocardin gene expression and activity and in switching smooth muscle cell phenotype that mediates remodeling of arteries in diseases like pulmonary hypertension and metabolic syndrome.
A Chemical Toolbox for Lysine Deacetylases
Authors: Schwarzer Dirk
Published: June 2021

Lysine acetylation is a well‐known post‐translation modification of proteins, which was discovered in the 1960s. Histone proteins and in particular their N‐terminal tail regions are among the most prominent targets of this modification. Histones package DNA into chromatin and lysine acetylation serves as an important regulator of chromatin condensation and accessibility and thereby gene expression. Recent proteome‐wide investigations have uncovered that lysine acetylation is not limited to histones but constitutes one of the most abundant protein modifications in nature with 3600 acetylation sites identified in the human proteome. Subsequent biochemical investigations have linked lysine acetylation to the regulatory networks of several cellular processes including signal transduction, apoptosis and metabolism. Lysine acetylation is catalyzed by lysine acetyltransferases and erased by lysine deacetylases. Especially the latter class of enzymes has gained considerable attention because lysine deacetylases have been identified as promising drug targets. However, biochemical research is hampered by the poor accessibility of these enzymes and as a result the overall knowledge about the substrate selectivity of lysine deacetylases is poor. In this talk recent developments and applications of chemical tools for probing the activity and specificity of lysine deacetylases will be presented.
Probing Protein Intrinsic Disorder and Allostery Modulation at Single‐Molecule Resolution
Authors: Deniz Ashok
Published: June 2021

The biophysics of protein intrinsic disorder is being recognized to be a critical component of biology. However, disordered proteins and their interactions can be dynamic and complex, making them less amenable to studies by traditional tools of biophysics and structural biology that can mask information due to averaging over an ensemble. We develop and apply novel tools that can be used to study molecules one at a time, thus revealing hidden structural and dynamic properties of these important protein systems. We will discuss examples of our studies that reveal new information about protein disorder and its role in modulating protein binding cooperativity and allostery, features that are important for function and misfunction in cells. Overall, our results highlight strengths of single‐molecule techniques to study the complex biophysics of structure and interactions of disordered protein systems.
Characterizing Heterogeneous Cellular Responses to Polymethoxyflavones Using Raman Microscopy
Authors: Zhang Hua , He Lili , Xiao Hang
Published: June 2021

To understand how individual cells respond to perturbations is a challenge as much of our knowledge is based on ensemble measurements. While cellular heterogeneity cannot be ignored, a practical analytical tool for analyzing and interpreting cellular heterogeneity is needed. Herein, we report the use of Raman Spectroscopy to characterize heterogen­eous cellular responses of human colon cancer cells to polymethoxyfla­­vones (PMFs), a group of anti‐cancer flavonoids found in citrus fruits. Raman spectroscopy is a molecular vibrational spectroscopy can measure chemical signatures of a single cell noninvasively. In this study, the cells were treated with PMFs, i.e. nobiletin (NBT) and 5‐demethylnobiletin (5DN) for 24, 48 and 72 hours, and both the floating and the attached cells were collected for Raman measurement. Heterogeneity of their Raman spectra was analyzed by principal component analysis (PCA). The results showed that Raman Spectroscopy could clearly characterize the difference between floating and attached cells under different treatments and at different treatment time. The biochemical profiles of floating cells are more heterogeneous in protein compared with those of the attached cells, indicating the different sensitivity and behavior of individual cells responding to NBT or 5DN. This phenomenon was less pronounced when cells were treated for longer period of time (72 hours vs. 48 hours). Our findings demonstrated that Raman microscopy offers a reliable method for characterizing individual cells, which can be used to investigate the heterogeneous responses of cell population to environmental stimuli.
Imaging the Dynamics of Bone Elongation: Integrating Macro‐ and Micro‐scale Processes at the Organ Level in situ
Authors: Farnum Cornelia
Published: June 2021

Knowledge of variables associated with endochondral ossification has been based on synthesizing observations from multiple sources: clinical abnormalities of long bone growth; physiologically‐based whole animal experiments; chondrocytic biochemistry/physiology; and molecular analyses including murine models with genetic manipulations. Molecular approaches have been particularly robust at enumerating the panoply of players potentially involved, but the challenge remains of understanding the role of these players in an organ level system of an animal growing through time. Live animal imaging offers the potential to maintain the organ‐level environment of the growth plate for functional analysis of the real‐time complexity of movement of potential regulatory molecules within the extracellular matrix, communication links among chondrocytes, and interactions of chondrocytes with cells in adjacent bone, vasculature, and perichondrium. This approach can be extended by manipulating other complex variables known to affect the rate and extent of postnatal bone elongation such as nutritional status, exercise status, or any of a variety of genetic changes resulting in compromised elongation and abnormal stature. Thus imaging in situ has the potential to return analyses of growth plate regulation to its organ‐level environment and to answer questions of possible value for therapeutic discovery.
Selenium Status Biomarkers and Selenium Requirements in the Turkey (M. gallopavo)
Authors: Taylor Rachel , Sunde Roger
Published: June 2021

Glutathione peroxidase (Gpx) activity has been used extensively to determine selenium (Se) status and requirements. The identification of the full selenoproteome and the use of transcriptomics offer additional biomarkers for assessment of Se status. We recently reported the Se requirement for the turkey based on Gpx1 and Gpx4 activity was 0.3 mg Se/g diet (vs. 0.2 mg/g NRC 1994), and that both Gpx mRNAs were reduced by Se deficiency (Exp Biol Mol 235:23). To determine Se status in today's rapidly growing commercial turkeys using both traditional and mRNA biomarkers, we fed day‐old male poults (n=50) a Se‐deficient diet (0.005 mg/g) supplemented with 10 graded levels of Se (0‐1.0 mg/g) for 28 d. There was no significant effect of Se on growth, although deficient (–Se) poults averaged 533±134 g vs. 769±202 g in 0.4 mg/g poults. Se response curves and plateau breakpoints (BP) were used to determine the impact of deficiency and minimal Se requirements. Plasma Gpx3 activity (n=5/trt) in –Se poults fell to <2% of Se‐adequate levels (+Se), showing our birds were Se deficient. The BP for plasma Gpx3 activity was 0.22 mg/g. Gpx1 in red blood cells fell to 34% of +Se and reached a BP at 0.25 mg/g. Initial analysis (n=3/trt) showed liver Gpx1 and Gpx4 activity in –Se poults fell to 4 and 6% of +Se, respectively, and reached BP at 0.28 and 0.14 mg/g, respectively. qRT‐PCR of total RNA (n=3/trt) showed –Se liver Gpx1 and Gpx4 mRNA levels fell to 32 and 17%, respectively, of +Se, with both BPs at 0.05 mg/g. Turkey Se requirements based on Gpx activity are ~3x higher than in rats but Gpx1 mRNA‐based requirements are the same as in rats. This study shows Gpx4 liver mRNA and activity are highly regulated by Se status in turkeys, in contrast to rodents, and that the turkey minimal Se requirement is 0.3 mg Se/g. Support: WIS 1829 and UW Se Nutrition Research Fund
Translation Initiation Signaling in Myotubes from Normal and Low Birth Weight Neonatal Pigs
Authors: Chen Ying , Zhu Haibo , El‐Kadi Samer
Published: June 2021

Skeletal muscle growth is impaired in low birth weight (LBWT) compared to normal birth weight (NBWT) neonates. Our previous studies showed that skeletal muscle protein synthesis is lower and IGF‐І receptor expression is higher in LBWT than NBWT neonatal pigs. The objective of the current study was to investigate the effect of IGF‐І stimulation on the regulation of translation initiation signaling in primary myotubes from LBWT and NBWT neonatal pigs. Primary satellite cells were isolated from Longissimus dorsi muscle of male LBWT and NBWT pigs (n = 4). Cells were allowed to proliferate to ~80% confluence, and differentiate to form myotubes. At 2.5 day of differentiation, myotubes were treated with IGF‐І at physiological levels (0, 25 and 50 ng/ml) after fasting in serum‐free media for 3 h. Myotubes were harvested in lysis buffer, and the expression of translation initiation signaling proteins were measured by Western blotting. IGF‐І stimulation significantly increased protein kinase B (PKB/Akt), eukaryotic initiation factor 4E binding protein (4EBP1) and ribosomal protein S6 kinase beta‐1 (S6K1) phosphorylation in myotubes from LBWT and NBWT pigs (P < 0.05). However, total S6K1 expression was significantly lower (P < 0.05) and 4EBP1 phosphorylation was higher (P = 0.09) in myotubes of LBWT compared with NBWT pigs. Our data suggest IGF‐І stimulation does not differentially regulate translation initiation signaling in myotubes from LBWT and NBWT pigs. S6K1 expression is reduced in primary myotubes from LBWT pigs, which likely indicates impaired translation initiation signaling in myotubes from LBWT compared with NBWT pigs.
Reduction of Nitrosative Stress by Vitamin C: a New Approach for Mitigation of Doxorubicin‐induced Cardiomyopathy
Authors: Akolkar Gauri , Bagchi Ashim , Mandal Soma , Jassal Davinder , Singal Pawan
Published: June 2021

Background

Despite the effectiveness of Doxorubicin (Dox) as an anti‐cancer drug, the dose dependent cardiotoxic side effects are serious life threatening concerns associated with its use. In addition to the oxidative stress, there is increasing evidence of involvement of nitrosative stress in Dox‐induced cardiotoxicity. Previously, Vitamin C (Vit C) has been shown to reduce oxidative stress and improved survival of Dox‐treated cardiomyocytes. However there is no evidence on the effect of Vit C on nitrosative stress. The objective of this study was to investigate the effect of Vit C on nitrosative stress in Dox treated cardiomyocytes.

Methods and Results

Cardiomyocytes isolated from adult Sprague‐Dawley rats were treated with predetermined doses of Vit C (25 µM), Dox (10 µM) or Vit C (25 µM) + Dox (10 µM) for 24 hours. Dox treatment increased levels of nitrate in cardiomyocytes as well as released in media leading to increased production of peroxynitrite. Vit C significantly reduced the levels of cellular and released nitrate, cellular peroxynitrite as well as the nitrosylation of proteins caused by Dox. Dox induced increase in Nitric oxide synthase (NOS) activity in cardiomyocytes was blunted by Vit C treatment. Western blot analysis revealed an upregulation of inducible NOS (iNOS) and phosphorylation of inhibitory site of endothelial NOS (eNOS) by Dox. In contrast Vit C downregulated iNOS and phosphorylated activating site of eNOS. Dox induced activation of apoptosis was prevented by Vit C.

Conclusion

These results provide a hope and rationale for use of Vit C as an adjuvant therapy to mitigate cardiotoxic side effects of Dox by attenuating oxidative and nitrosative stress.

Support: CIHR
Visualization of Dietary Patterns
Authors: Krebs‐Smith Susan , Wilson Magdalena , Reedy Jill
Published: June 2021

Diet quality indices, such as the Healthy Eating Index 2010 (HEI‐2010), include numerous components to capture the multidimensionality of diets, but typically only the total index score indicative of overall diet quality is presented. This project examined the merit of radar graphs for depicting multidimensional dietary patterns and how those patterns vary in the US population over time and by different characteristics.

National Health and Nutrition Examination Survey (NHANES) dietary intake data were used to estimate HEI‐2010 component scores for the US population, over time (from 1999/2000 to 2009/2010), by socioeconomic status (i.e. poverty income ratio), and by age and sex categories. Each component score was scaled from 0‐100% and plotted. A perfect HEI‐2010 score, representing a diet that conforms to the 2010 Dietary Guidelines for Americans, would be depicted as a line around the border of the radar plot.

Points on all axes of the radar plots show the relative scores for the components, and the overall shape provides an indication of the dietary pattern, with larger shapes generally indicating higher dietary quality. The spacing between different groups' points on each axis shows the difference in quality for that component between those groups. The plots show that some components—such as whole fruit and dairy—vary greatly, while others—such as sodium and total protein foods—vary less. The components that vary greatly drive the differences in overall diet quality between groups.

Radar plots are a useful tool to conceptualize dietary patterns. They allow for visualization of differences in patterns between groups, and trends in dietary patterns over time. However, as the shape of the patterns depends on the order of the components presented on the plot, it is important for the order of components to be standardized across analyses using this tool.