bluuebunny/crossref_metadata_2025 huggingface dataset:
Live tissue imaging of organic cation and anion transport at the blood‐CSF barrier reveals specific transporter function and distinct transcellular pathways
Authors: Hu Tao , Zha Weibin , Duan Haichuan , Wang Joanne
Published: November 2023

Formed by the choroid plexus (CP) epithelial cells, the blood‐cerebrospinal fluid (CSF) barrier plays an active role in removing neurotoxins, drugs, and metabolites from the brain. Several organic cation and anion transporters, including the plasma membrane monoamine transporter (PMAT), organic anion transporter 1 and 3 (OAT1/3), and multidrug resistance‐associated protein 1 and 4 (MRP1/4) have been reported to be expressed in CP, but their relative expression and specific function at the blood‐CSF barriers have not been well characterized at the tissue level. The goal of this study is to quantify the expression of organic cation and anion transporters in CP tissues and determine their specific roles in organic cation or anion transport by live tissue imaging using fluorescent substrates. In both human and mouse CPs, real‐time PCR analysis showed highest mRNA expression of PMAT followed by MRP1, OAT3 and MRP4. CP also expresses several drug metabolizing enzymes including the monoamine oxidases (MAOs), catechol‐O‐methyltransferase (COMT), UDP‐glucuronosyltransferases (UGTs) and sulfotransferases (SULT). In CP isolated from wildtype (wt) mouse, the fluorescent organic cation probe IDT307 is transported into CP cells at the apical (CSF‐facing) membrane and is highly accumulated in CP cells with a large storage in mitochondria. There was little efflux at the basolateral membrane. In contrast, IDT307 uptake and intracellular accumulation is attenuated by ~3–4 folds in CPs isolated from Pmat knockout mice. Interestingly, in wt CPs, the organic anion probe fluorescein‐methotrexate (FL‐MTX) is rapidly taken up and efficiently excreted into the capillary side with little intracellular accumulation. MK571, a potent inhibitor of MRP1/4, abolished basolateral efflux of FL‐MTX, resulting in high accumulation within CP cells. Together, our study in live CP tissue demonstrates an important role of PMAT in apical uptake of organic cations and a crucial function of MRP1/4 in basolateral efflux of organic anions. Based on these studies, we propose distinct molecular and cellular pathways for organic cation and anion transport at the blood‐CSF barrier.

Support or Funding Information

This study is supported by National Institutes of Health Grant GM066233.
Identification of transport systems for fluorescent bile salt derivatives
Authors: Stieger Bruno , Leuenberger Michele , Stephanie Häusler , Lochner Martin
Published: November 2023

Organic anion transporters of hepatocytes are mediating uptake of bile salts as well as drugs into hepatocytes. Consequently, transport of endogenous substrates and drugs by these transporters is routinely investigated during drug development as well as in basic research studying physiology and pathophysiology of bile formation. In order to facility transport measurements, fluorescent transporter substrates are desirable. We therefore aimed to develop novel fluorescent substrates for the bile salt transporters sodium‐taurocholate cotransporting polypeptide (NTCP) and bile salt export pump (BSEP) as well as for the hepatocellular organic anion transporting polypeptides (OATPs). Three fluorescent analogs of cholylethylenediamine amides (Cen) were synthesized with the fluorescence moiety coupled to the side chain: CenNBD (Cen with NBD), CenDansyl (Cen with dansyl) and CenPB (Cen with pacific blue). Uptake experiments were performed using CHO cells stably transfected with NTCP, OATP1B1, OATP1B3 or OATP2B1 and insect cell vesicles expressing BSEP with standard substrates as positive control and with fluorescent bile salts. All fluorescent bile salts inhibited the investigated OATPs in a concentration dependent manner. All three fluorescent bile salts were substrates of OATP2B1 and to some extent of OATP1B1 and OATP1B3. In contrast, only CenDansyl was a substrate of NTCP. This bile salt derivative is an inhibitor of BSEP, but not a substrate. Our data confirm that a chemical modification of the side chain of bile salts has a considerable impact on the transporter selectivity and on the transport efficiency of bile salt derivatives.

Support or Funding Information

SNF National Center of Competence in Research TransCure, University of Bern, Switzerland
Prophylactic and therapeutic effect of human placenta‐derived mesenchymal stem cells on radiation enteropathy with discovery of biomarkers predicting favorable response
Authors: Han Young‐Min , Park Jong‐Min , Hahm Ki Baik
Published: November 2023

Radiation enteropathy is a common complication in patients with abdomino‐pelvic cancer, but no treatment has yet been established. Stem‐cell therapy may be a viable therapeutic option based on the following observations: i) intestinal stem‐cells are highly vulnerable to ionizing‐radiation (IR) and ii) Stem‐cells loss explains its intractability. Here, we investigated the prophylactic and therapeutic efficacy of human‐placenta‐derived‐mesenchymal‐stem‐cells (hPDSCs) against radiation enteropathy and identified biomarkers that may predict a favorable response to cell therapy. hPDSCs improve the outcome of mice induced with either radiation enteropathy or lethal radiation syndrome. hPDSCs exerted inhibitory actions of inflammatory cytokines. The re‐establishment of epithelium homeostasis was completed with increasing endogenous restorative processes as assessed with increased levels of proliferative markers in hPDSCs group, whereas an inhibition of IR‐induced apoptosis was observed following hPDSCs treatment. The preservation of cells expressing lysozyme, and Musashi‐1 were noted in the hPDSC treatment group. Label‐free quantification was used to identify biomarkers which predict favorable responses after hPDSCs‐treatment finally GST‐(mu) and Peroxiredoxin‐2 were validated as biomarkers predicting favorable response following treatment with hPDSCs. The fact that administration of hPDSCs was effective to mitigate radiation enteropathy suggests that hPDSCs may be a useful prophylactic and therapeutic cell therapy for radiation enteropathy.
The Role of VE‐PTP Scaffold in Stabilizing the Adherens Junction
Authors: Juettner Vanessa , Dan Arkaprava , Leckband Deborah , Komarova Yulia , Malik Asrar
Published: November 2023

The Vascular Endothelial Protein Tyrosine Phosphatase (VE‐PTP) is an endothelial‐specific phosphatase that dephosphorylates multiple proteins including Vascular Endothelial (VE)‐cadherin, the main adhesion protein of adherens junctions (AJ). However, the role of VE‐PTP in stabilization of VE‐cadherin adhesion complexes remains unclear. VE‐cadherin establishes adhesion events through trans interactions with opposing VE‐cadherin molecules at AJs. In resting endothelial monolayers, it undergoes continues exchange between junctional and cytosolic pools. Using VE‐cadherin tagged with the photoconvertable protein Dendra2, we have demonstrated that VE‐PTP regulates steady‐state kinetics of VE‐cadherin at AJs in a phosphatase‐independent manner. Overexpression of wild‐type (WT) or phosphatase “dead” VE‐PTP mutant similarly decreased the VE‐cadherin dissociation rate from AJs as compared to control cells expressing empty vector. In agreement with these results, VE‐PTP depletion increased VE‐cadherin dissociation rate from AJs. Furthermore, treatment of the cells with the VE‐PTP phosphatase inhibitor, AKB‐9785, did not alter VE‐cadherin dynamics suggesting that VE‐PTP promoted stability of VE‐cadherin adhesion by decreasing VE‐cadherin dissociation rate in a phosphatase‐independent manner. Because Rac1 stabilizes AJs by reducing mechanical tension across VE‐cadherin adhesion, we analyzed junctional activity of Rac1 as well as the tension applied to VE‐cadherin using respective FRET biosensors. We observed that overexpression of VE‐PTP caused both Rac1 activation at AJs and reduction of tension across VE‐cadherin adhesion. Consistent with these results, VE‐PTP knockdown decreased Rac1 activity at AJs and increased stress at AJs determined using micropillar arrays. Cumulatively, our data suggest a scaffold role of VE‐PTP in regulating Rac1 activity at AJs. Future studies will further investigate the molecular mechanisms by which VE‐PTP activates Rac1.

Support or Funding Information

Funding: National Institutes of Health grant R01 HL103922 to Y.A. Komarova, and T32 and RO1 HL45638 to A.B. Malik.
Critical Role of Vitamin D3 in Helicobacter pylori Eradication in Stomachs
Authors: Hu Wei
Published: November 2023

Backgrounds and objectives

Helicobacter pylori (H. pylori) establishes a new and important field in clinical microbiology, as the bacterium was found to be a major cause of several severe gastric disorders, including chronic gastritis and peptic ulcers. It is also a risk factor for adenocarcinoma and gastric lymphoma. Antibiotic treatment is adopted as the first therapeutic approach for H. pylori eradication. However, with the prevalence of antibiotics usage, multidrug resistance (MDR) is found to be increasingly common and also critical in clinics in the past decades. Therefore, new strategies of drug treatment are urgently needed for patients with H. pylori infection. This study aimed at demonstrating the critical role of vitamin D3 in H. pylori eradication.

Methods

PCR and immunostaning assay were used to determine the intracellular H. pylori level. Western blot assay was used to determine the autophagy‐related proteins level. Acridine Orange and GFP‐mCherry‐LC3 plasmid transfection were used to measure lysosomal acidification.

Results

We found a promising effect of vitamin D3 in clearing H. pylori infection both in gastric epithelial cells and in animals. The action was found to be independent on cathelicidin expression which was activated by vitamin D3 and shown to have anti‐bacterial action. Importantly, it was demonstrated that vitamin D3 could revitalize the degradation function of lysosomes which was adversely affected by H. pylori in cells after infection. This restoring effect was accomplished by lysosomal acidification enhancement. Moreover, we also found that the therapeutic action was not acting through vitamin D receptor, instead it was mediated by a separate receptor, the PDIA3 inside the cell, following by activating the calcium channel TRPML3 in lysosomes, and the degradation function can be restored by vitamin D3 through PDIA3 receptor.

Conclusions

Our studies revealed a novel and unique antibacterial pathway for Vitamin D3, which is different from the mechanisms of action for traditional antibiotics. Therefore, vitamin D3 could be a new therapeutic agent for bacterial infection and in particular those MDR bacteria in the future.

Support or Funding Information

This study was partly supported by the General Research Fund from the Hong Kong Research Grant Council (CUHK 463613) and the Health and Medical Research Fund from the Food and Health Bureau (13120062) in Hong Kong and the National Natural Science Foundation of China (No. 81473269).
Obesity‐Induced Mouse Prostatic Dysfunction is Improved by Chronic Treatment with Resveratrol: Implication of Oxidative Stress and Neuronal Growth Factor
Authors: Calmasini Fabiano Beraldi , Oliveira Mariana Gonçalves , Alexandre Eduardo Costa , Silva Fabio Henrique , Tavares Edith Bastos Gandra , Antunes Edson
Published: November 2023

Introduction

The worldwide incidence of obesity has increased dramatically in the past 25 years. Clinical studies indicate a strong correlation between obesity and genitourinary tract diseases, being benign prostatic hyperplasia (BPH) one of the most common prostatic disorders affecting obese individuals1,2. Recently, increased oxidative stress has been shown to play a pivotal role in BPH pathogenesis/progression3. In the present study we explored the effects of the polyphenol resveratrol, an antioxidant drug, on obesity‐induced prostatic dysfunctions.

Methods

Male C57BL6/JUnib mice, fed for 12 weeks with either a standard chow or high‐fat diet to induce obesity were treated daily with resveratrol (100 mg/kg/day, gavage) from weeks 10–12 of diet. Concentration‐response curves to phenylephrine (PE; α1‐adrenoceptor agonist) and potassium chloride (KCl; depolarizing agent) as well as neurogenic contractions produced by electrical‐field stimulation (EFS; 1–32 Hz, 50 V, 10 s) were obtained. Stereological analysis, reactive‐oxygen species (ROS) levels, RT‐PCR for neuronal growth factor (NGF) and NADPH subunit gp91phox were all performed in the prostatic tissues in all groups.

Results

A significant increase (P<0.01) in the prostate smooth muscle contractions (PSM) to PE and KCl were observed in obese group compared with control animals. Likewise, EFS‐induced PSM contractions were higher in obese animals in all frequencies employed. Resveratrol fully restored the enhanced PSM contractions to PE, KCl and EFS in obese group. ROS levels were 87% higher (P<0.001) in prostate from obese mice. Moreover, mRNA expression for gp91phox was 57% higher (P<0.05) in prostate of obese mice. Resveratrol restored the augmented ROS production and gp91phox mRNA in obese mice, without affecting these parameters in the control group. In prostate from obese mice, the stereological analysis showed epithelial polyps formation and reduced lumen area whereas stromal and smooth muscle cells layer remained unchanged. A significant increase by about of 70% (P<0.01) in mRNA expression for NGF was also found in prostate of obese mice. Resveratrol significantly restored the NGF mRNA expression, and attenuated the epithelial alterations, without affecting the reduced luminal area.

Conclusion

A two‐week treatment with resveratrol is capable to reverse the prostatic dysfunctions in obesity mice. Resveratrol may be a good pharmacological option to prevent obesity‐associated prostatic dysfunctions.

Support or Funding Information

Fundação de Amparo à Pesquisa do Estado de São Paulo ‐ FAPESP (2016/01178‐6)
Drugging the Gut Microbiome for the Treatment of Alcohol‐Induced Liver Injury
Authors: Helsley Robert N. , Schugar Rebecca C. , Ferguson Daniel , Gromovsky Anthony D. , Neumann Chase , Wang Zeneng , Allende Daniela , Nagy Laura E. , Hazen Stanley L. , Brown Jonathan M.
Published: November 2023

Objectives

Sustained alcohol abuse initiates a progressive disease pathogenesis involving simple steatosis, steatohepatitis, fibrosis, cirrhosis, and in some cases hepatocellular carcinoma. There is mounting evidence that microbes resident in the human intestine represent a key transmissible factor promoting inflammatory diseases including alcoholic liver disease (ALD). However, mechanisms by which gut microbiota synergize with excessive alcohol intake to promote ALD are poorly understood. We have recently revealed a metaorganismal pathway where nutrients in high fat foods can be metabolized by gut microbes to generate trimethylamine (TMA), which is subsequently metabolized by the host enzyme flavin‐containing monooxygenase 3 (FMO3) to produce the pro‐atherogenic metabolite trimethylamine‐N‐oxide (TMAO). Hepatic FMO3 mRNA levels are suppressed in alcoholics and breath levels of TMA correlate with the severity of alcoholic hepatitis. Thus, this study investigates whether pharmacological inhibition of gut microbe‐derived TMA production can blunt ethanol‐induced liver injury

Methods and Results

We first determined if ethanol feeding would alter circulating TMA and TMAO levels in mice. Acute (6% vol/vol Lieber DiCarli) and chronic (25 days Lieber DiCarli) isocaloric pair‐ and ethanol‐feeding elicited increases in both TMA and TMAO levels. We then employed a novel approach to block TMA production by using a small molecule inhibitor (CC08) targeting the gut microbial TMA‐producing enzyme CutC/D. CC08 treatment effectively abolished ethanol‐elicited increases in TMA and TMAO. In parallel, CC08 treated mice were completely protected against ethanol‐induced hepatic fat deposition, hepatocellular inflammation, and elevated alanine aminotransferase levels. Collectively, these results suggest inhibiting gut microbial production of TMA can blunt ethanol‐induced liver injury.

Conclusions

Our results establish the TMA/TMAO pathway as an important regulator of ethanol‐induced liver injury. Inhibition of TMA production by utilizing small molecule inhibitors targeting the TMA lyase, such as CC08, may represent a novel therapeutic approach to combat ethanol‐induced liver injury.

Support or Funding Information

P50 (AA024333‐01)
Dendrobium Officinale Extract Increases the Expression of Aquaporin 5 through Activation of ERK and JNK in Lacrimal Gland Cells
Authors: Zeng Qiang , Chun‐Hay KO , Siu Wing‐Sum , Wong Chun‐Wai , Hu Jiang‐Miao , Lau Clara Bik‐San , Leung Ping‐Chung
Published: November 2023

Dendrobium officinale has been used as a healthy food and a source of nutrients for thousands of years in China. It is traditionally used to nourish stomach, maintain body fluid production and treat thirst, sore throat and blurred vision. Dendrobium officinale and its formulations are used to promote body fluid production in dry eye disease or hyposalivation of patients after head and neck radiotherapy. Recently, polysaccharides isolated from Dendrobium officinale have been demonstrated to protect salivary gland and stimulate expression of aquaporin 5 (AQP5) in labial glands from Sjögren's syndrome patients. However, the underlying mechanism of promotive effect of Dendrobium officinale on body fluid secretion is seldom reported. Therefore, our study aimed to determine whether Dendrobium officinale aqueous extract could increase the expression of AQP5 through activation of MAPKs pathways in primary cultured lacrimal gland cell. Primary lacrimal gland cells were isolated from male SD rats and characterized by immunofluorescence. Cell viability and cell proliferation were evaluated by MTT assay and BrdU assay, respectively. The results showed aqueous extract of Dendrobium officinale (DOW) increased the lacrimal gland cell viability, but not in cell proliferation. The mRNA levels of β‐hexosaminidase A (HexA) and AQP5 were increased after DOW treatment for 4 hours by RT‐PCR. Protein levels were measured by western blotting. The increase of AQP5 expression was greater after 24‐hour and returned to its ordinary level by 48‐hour treatment at dose of 800 μg/ml. Moreover, DOW stimulated phosphorylation of ERK and JNK with 800 μg/ml treatment and peaked after 30 min stimulation. In conclusion, we firstly presented evidence that Dendrobium officinale aqueous extract increased the expression of aquaporin 5 through activation of ERK and JNK pathways in lacrimal gland cells. Our data has provided a solid background for Dendrobium officinale to be developed as an alternative or adjuvant intervention in dry eye disease.
Role of Hypoxia‐Inducible‐Factor‐2α in Transcriptional Regulation of Heme Oxygenase‐1 in Hypoxic Cardiomyocytes
Authors: Eadie Ashley L. , Allwood Melissa A. , Platt Mathew J. , Huber Jason , Trivedi Purvi C. , Madhu Malav N. , Pulinilkunnil Thomas C. , Simpson Jeremy A. , Brunt Keith R.
Published: November 2023

INTRODUCTION

The heme metabolism pathway is a novel therapeutic target in the management of heart failure, with cytoprotection mediated through induction of heme oxygenase‐1 (HMOX1). However, translation of HMOX1‐targeted therapeutics to the clinic has not occurred. Understanding of heme bioavailability regulation in cardiomyocytes, particularly during hypoxia or heart failure, is incomplete. The molecular response to hypoxia is largely regulated by hypoxia inducible transcription factors (HIFs). HIF1α transcriptionally regulates HMOX1 in acute, severe hypoxia but the role of HIF2α in heme metabolism is unknown. A detailed understanding of the differences in molecular regulation between acute or chronic and severe or moderate hypoxia in cardiomyocytes is required.

HYPOTHESIS

Hypoxia‐inducible factor 2α (HIF2α) contains heme‐regulatory motifs that are sensitized by heme bioavailability at various degrees of hypoxia in cardiomyocytes to regulate the acute expression of HMOX1 in vitro and in experimental models of heart failure in vivo.

METHODS

Acute myocardial infarction (AMI)‐induced heart failure was modeled through permanent surgical ligation of the left anterior descending coronary artery in mice. Hypertensive heart failure was modeled through transverse aortic constriction (TAC). Expression of heme regulatory enzymes and hypoxia inducible factors were measured via Western Blot, qPCR and heme content assay. In vitro, hypoxia was established by incubating H9C2 and neonatal rat cardiomyocytes in an adjustable‐oxygen chamber. HIF1α and HIF2α were knocked down or overexpressed through adenoviral vectors. To investigate the role of heme bioavailability in HIF2α sensitization, cells were incubated with the heme surrogate, hemin.

RESULTS

In vivo, heme levels were increased in both AMI and TAC‐induced heart failure progressively, however not due to increased heme synthesis enzymes. In vitro, HMOX1 was increased by HIF2α more than by HIF1α overexpression. This response was synergistically increased in heme‐replete hypoxia with HIF2α, but not HIF1α, overexpression.

CONCLUSIONS

We have shown for the first time that HIF2α is a novel regulator of HMOX1. Further understanding of heme homeostasis in the heart is necessary to optimize therapeutic targeting of the heme metabolic pathway in heart failure. Further, we show that HIF2α has a distinct role from HIF1α as it is sensitive to heme bioavailability and not an exclusive oxygen sensor.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada
A transgenic mouse model to study a novel PRKAG2 mutation (K475E) causing infantile‐onset hypertrophic cardiomyopathy
Authors: XU YANCHUN , Liu Hong , Phornphutkul Chanika , Hardie Grahame , Dudley Samuel C , Padbury James F , Tseng Yi‐Tang
Published: November 2023

Background

PRKAG2 encodes the γ2 subunit isoform of AMPK, a heterotrimeric enzyme with major roles in regulation of energy metabolism in response to cellular stress. Mutations in PRKAG2 have been implicated in hypertrophic cardiomyopathy (HCM). We identified a novel PRKAG2 mutation in a neonate with prenatal onset of HCM noted on a 27‐week prenatal ultrasound. Molecular testing for HCM revealed a de novo PRKAG2 mutation (K475E). We have demonstrated that overexpressing the K475E mutant in vitro leads to activation of cell growth pathway. We also have demonstrated that K475E mutation led to increases in cell size and area which can be reversed by rapamycin.

Objective

To developed transgenic mouse models to confirm K475E‐induced cardiac phenotype, determine the downstream signaling pathways involved, and test targeted therapy for K475E‐induced HCM using rapamycin.

Methods

Human WT and K475E DNA constructs were inserted into a pBluescript vector with the mouse α‐myosin heavy chain promoter. Transgenic mice with the cardiac‐specific overexpression of WT (TgWT) or the K475E mutation (TgK475E) were established by pronucleus microinjection. Survival curve and cardiac hypertrophy were examined and cardiac function changes were monitored by echocardiography.

Results

Male TgK475E mice were prone to sudden death with only 50% survived by 13‐week old. Hypertrophy in TgK475E mice was confirmed by their significant increases in heart weight/body weight and heart weight/tibial length ratios versus the non‐transgenic control and TgWT mice. Histology revealed large vacuoles and high glycogen content in TgK475E myocytes throughout the ventricles. Echocardiography confirmed that LV mass dramatically increased in TgK475E mice starting from 5 weeks of age. At 6‐week old, ejection fraction (EF) was more than 70% in TgK475E mice, which then decreased to less than 30% by 18‐week old. Fraction shortening (FS) in TgK475E mice showed similar trend. In contrast, EF and FS in non‐transgenic control and TgWT mice were not significantly changed. Compared to nontransgenic control and TgWT mice, Vd, Vs, LIVD (d) and LIVD (s) were all significantly increased in TgK475E mice starting at 7‐week of age. 4‐week Rapamycin treatment dramatically decreased the ratios of heart weight/body weight and heart weight/tibia length in TgK475E mice.

Conclusion

These results indicate the transgenic mouse model reflects the early onset phenotype of the K475E mutation‐induced HCM. Targeted therapeutic strategy with mTOR inhibition is partially effective and should be studied further.

Support or Funding Information

This study was supported by NIH P30GM114750 (J.P., Y.‐T.T.) and Oh‐Zopfi Research Award, Women & Infant's Hospital (Y.‐T.T.). We thank the COBRE in Perinatal Biology Core and Kilguss Core Facility for the excellent molecular and microscopy supports.
Spontaneous Dystrophic Cardiac Calcinosis in C.B‐17 SCID Mice
Authors: Raghunathan Suchi , Reynolds Corey L. , Stewart M. David , Schwartz Robert J. , McConnell Bradley K
Published: November 2023

Animal models have a pivotal role in biomedical research and contribute significantly to the study of human disease. C.B‐17 SCID mice which are homozygous for prkdscid mutation and have BALB/c background, have been used to support research in cancer, diabetes, obesity, cardiovascular diseases and many other areas. In the current study, we report severe cardiac calcinosis in C.B‐17 SCID male mice at 10 weeks of age, particularly the right ventricular myocardium. Dystrophic cardiac calcinosis is a characteristic of certain inbred strains of mice of which BALB/c background mice exhibit more susceptibility. It is characterized by myocardial injury, calcification and necrosis of the tissue. Here, we analyzed the ECG and echocardiogram of C.B‐17 SCID mice from two different colonies; Germantown, NY colony and Cambridge City, IN colony of Taconic Biosciences. Histological analysis was used to characterize the calcinotic lesions and fibrosis. At 10 weeks of age, 61% of the total C.B‐17 SCID mice from both colonies exhibited significant ventricular calcinosis, predominantly on the right ventricle. The frequency of calcinosis was more than 50% in the Cambridge City mouse colony and 25% in the Germantown mouse colony. The ECG recordings of unanesthetized mice did not exhibit any significant differences between calcinotic and non‐calcinotic mice from either colony. Cardiac function wall dimensions, derived from cardiac echocardiograms, also did not exhibit any significant difference between calcinotic and non‐calcinotic mice from either colonies. However, histology showed necrosis and calcium deposits within the myocardium. In conclusion, C.B‐17 SCID mice exhibit severe dystrophic cardiac calcinosis as early as 10 weeks of age. These strain of mice should be cautiously considered for other disease model studies where calcium homeostasis has a significant role to play.

Support or Funding Information

University of Houston
Post‐Infarct Remodeling in the Myocardium is Regulated by Hippo/YAP Signaling
Authors: Madhu Malav N. , Eadie Ashley L. , Aguiar Christie , Platt Mathew J. , Hassan Ansar , Simpson Jeremy A. , Brunt Keith R.
Published: November 2023

BACKGROUND

Ventricular remodeling is a complex process involving cell death, inflammation, fibrosis, cardiomyocyte hypertrophy. Recent studies show that Hippo/YAP signaling regulates cardiomyocyte growth and survival following ischemic injury in the myocardium. However, its role in ventricular remodeling is still poorly understood and may hold the potential for therapeutic targeting. In this study, we investigated the expression of Hippo/YAP signaling proteins in the mouse model of acute myocardial infarction.

METHODS

For acute myocardial infarction (AMI), mice were sedated with ketamine (50mg/kg) and xylazine (5mg/kg), intubated, ventilated, and maintained with 2% isoflurane. Through a left thoracotomy, the left coronary artery was ligated 1 to 2 mm below the border of the left atrial appendage. Animals were sacrificed after 4 weeks post AMI and hearts were harvested and frozen at −80°C. Tissues were homogenized and subjected to western blot analysis.

RESULTS

Immunoblot analysis of heart tissues show increased expression of β‐MHC, a protein commonly associated with ventricular remodeling and hypertrophy, in 4 week AMI hearts compared to sham. We report a significant increase in Hippo pathway components like Mst1, Mst2, pMob1 and YAP binding protein such as 14‐3‐3 in 4 week AMI hearts compared to sham hearts. On the other hand, YAP expression was reduced in 4 week AMI group compared to control group.

CONCLUSION

Collectively, our data suggest that the kinase cascade in Hippo/YAP pathway is activated resulting in YAP inactivation, which may contribute to ventricular remodeling following myocardial infarction.

Support or Funding Information

Natural Sciences and Engineering Research Council of Canada
Clofibrate, a Unique Fibrate that Augments Cardiac Contractility Through a PPARa‐independent Mechanism
Authors: Ponnapureddy Rakesh , Gill Tarana , Kim Seo‐Hyun , Mirza Omer , Stone Jessica , Anderson Jon , Touchberry Chad , Wacker Michael
Published: November 2023

Background

Fibrates are a group of synthetic peroxisomal proliferator‐activated receptor alpha (PPARα) agonists that have been in clinical use for several decades for their hypolipidemic properties. Clofibrate is the pioneer drug in the class of fibrates which have been shown to reduce triglyceride levels and elevate HDL cholesterol levels through PPARα agonism. More recently, non‐genomic actions of fibrates have received attention. In our previous experiments, we have demonstrated that fibrates induced acute relaxation of mouse aorta through non‐PPARα mediated mechanisms. Clofibrate, in particular, has been shown to inhibit Na+/K+ ATPase (NKA) activity in the rat kidney. It is well known that the NKA pump also plays an important role in maintaining calcium homeostasis in cardiomyocytes and NKA pump inhibition results in increased cardiac contractility. However, the direct effects of fibrates on cardiac muscle have not been tested. The objective of this study was to determine if clofibrate alters cardiac contractility independent of stimulation of PPARa.

Methods

Left ventricular muscle strips were isolated from 12–16 week‐old male wild type (WT) and PPARa null mice at 1 Hz in an oxygenated organ bath. Contractile waveforms were analyzed after treatment with clofibrate, clofibrate ester, gemfibrozil, and Wyeth 14,643 and also compared to the well‐known NKA pump inhibitor ouabain.

Results

We found that the effect of clofibrate on cardiac contractility in WT mice was concentration‐dependent with a 1.3 and 1.5‐fold increase above baseline at 2 and 20 μM concentrations, respectively (n= 7; P<0.05) and with no significant increase at 0.2 mM (P>0.05). The contractile response to clofibrate in PPARα deficient muscle strips was similar to WT with significant increases at 2 and 20 mM (n=7; P<0.05). Clofibrate ester, gemfibrozil and Wyeth 14,643, did not increase cardiac contractility (n=4; P>0.05). The change in contractile force of the muscle strips after administration of clofibrate was comparable to ouabain in WT mice (n=5; P<0.05).

Conclusions

Our findings show that clofibrate uniquely augments cardiac contractility through a PPARα‐independent mechanism which was comparable to the effect produced by the NKA pump inhibitor ouabain. Based on these results, there may be a new mechanism of action for clofibrate in cardiac contractility and could be explored as a novel heart failure treatment strategy.
Effect of Chronic Treatment With Zamicastat on Right Ventricle Pressure Overload in the Rat Monocrotaline Lung Injury Model of Pulmonary Hypertension
Authors: Igreja Bruno , Pires Nuno , Moser Paul , Soares‐da‐Silva Patrício
Published: November 2023

Pulmonary arterial hypertension (PAH) is defined, foremost, as a plexogenic arteriopathy with subtotal luminal obliteration that increases pulmonary vascular resistance and imposes a hemodynamic stress on the right ventricle (RV) leading to RV hypertrophy and failure that contributes to premature death. There is experimental and clinical evidence that supports a relation between PAH and the sympathetic nervous system (SNS), indicating that PAH can be mediated, at least partly, by SNS hyperactivation. A strategy for the modulation of sympathetic nerve function is to reduce the biosynthesis of norepinephrine (NE) by inhibiting dopamine β‐hydroxylase (DβH), the enzyme that catalyzes the conversion of dopamine (DA) to NE in sympathetic nerves. Here, we evaluated the effect of 18‐day oral treatment with the DβH inhibitor zamicastat on RV pressure overload in the monocrotaline (MCT)‐induced pulmonary hypertension (PH) model in the rat.

MCT increased RV systolic pressure (54.7±2.9 vs 32.3±1.0 mmHg, p<0.0001), mean RV pressure (23.9±1.7 vs 16.6±1.1 mmHg, p<0.001), and decreased heart rate (240.0±5.9 vs 285.8±7.6 beats/min, p<0.0001) in vehicle‐treated rats, as compared to pre‐MCT values. Zamicastat treatment prevented the MCT‐induced increase in mean RV pressure (change from baseline: +1.9±1.5 vs +7.4±1.8 mmHg, p<0.05). Likewise, the HR decrease was significantly attenuated in the zamicastat group as compared to the vehicle group (change from baseline: −14.3±9.7 vs −45.8±8.1 beats/min, p<0.05). Chronic zamicastat treatment decreased NE levels (480.5±43.9 vs 712.1±46.5 ng/mg protein, p<0.005) and increased DA levels (533.0±49.7 vs 12.3±2.3 ng/mg protein, p<0.0001) in adrenal gland homogenates of MCT‐treated rats, as compared to vehicle group demonstrating robust inhibition of DβH.

In conclusion, the DβH inhibitor zamicastat reverses heart rate and RV pressure changes, two hallmarks of PAH, in the rat MCT lung injury model.

Support or Funding Information

This work was supported by BIAL ‐ Portela & Ca, S.A.
The effects of Caffeic Acid Phenethyl Ester (CAPE) on oxidative stress and hypoxia‐induced cell damage
Authors: Martin Sarah , Castellano Andrew , Uzoaru Chijioke , Lim Jonathan , Carvis Nicholas , Wieczorek Peter , Barsotti Robert , Young Lindon H , Chen Qian
Published: November 2023

Oxidative stress has been implicated in pathogenesis of hypoxia and ischemia/reperfusion (I/R) injury. In previous studies, we have shown that the antioxidant CAPE exerted cardioprotection in an isolated rat heart I (30 min)/R (60 min) injury model. In this study, we further evaluated the effects of CAPE on oxidative stress and hypoxia‐induced cell damage. We evaluated the inhibition of absorbance in the phorbol 12‐myristate 13‐acetate (30 nM) induced superoxide production spectrophotometrically in isolated rat neutrophils via reduction of exogenous cytochrome C. We found that CAPE (0.5 μM–40 μM; n=4–13) reduced phorbol 12‐myristate 13‐acetate induced neutrophil superoxide release dose‐dependently from 29±3% to 95±2%. In a rat hind limb I (30 min)/R (60 min) model, blood hydrogen peroxide levels serves as an indicator of blood oxidative stress and was measured in real‐time via a hydrogen peroxide microsensor (100 μm) inserted into both femoral veins (one served as sham, the other as I/R). We found that in the control group, I/R significantly increased blood hydrogen peroxide levels to 2.1±0.8 μM relative to the sham limb at 60 minutes reperfusion when saline was given at the beginning of reperfusion (n=5). By contrast, CAPE when given at reperfusion (40 μM, n=5) significantly reduced blood hydrogen peroxide levels from 30 min reperfusion and throughout the rest of experiment (p<0.05). The relative change was −0.4±0.3 μM between the I/R and sham limbs at 60 minutes reperfusion. In H9C2 rat myoblast cells, cobalt chloride simulated hypoxia and induced cell damage. Tetrazolium, which can differentiate metabolically active and inactive cells/tissues, was used to measure cell damage by measuring absorbance at 450 nm. We found that incubation of cobalt chloride (800 μM, n=8) for 24 hr induced 55±5% cell death. By contrast, pretreatment of CAPE (0.5 μM–10 μM; n=1–3) for 24 hr dose‐dependently improved cell survival up to 30±9% relative to cobalt chloride treatment. In summary, this study suggests that CAPE attenuates oxidative stress from neutrophils and I/R, which may contribute to improvement in cell survival under hypoxia conditions.

Support or Funding Information

This study was supported by Division of Research and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.
Transcriptomic Analysis of the Stellate Ganglion After MI
Authors: Bayles Richard , Olivas Antoinette , Gardner Ryan , Burchard Julja , Habecker Beth
Published: November 2023

Survivors of a myocardial infarction (MI) remain at serious risk of sudden cardiac death due to lethal arrhythmias. It is known from studies in humans and dogs that electrical and structural remodeling of the stellate sympathetic neurons occurs following MI, including an increase in size and excitability. Increased excitability may be due to a variety of sources including increased dendritic field size, more synaptic inputs or altered ion channel expression to name a few. In an effort to identify neuronal genes regulated by MI, we have performed RNA sequencing (RNAseq) of RNA extracted from the stellate ganglia of mice 14 days after MI or sham surgery. Infarction was induced by 45 min occlusion of the left anterior descending coronary (LAD) artery, and sham operated animals underwent the same procedure with no occlusion. After 14 days, left ventricular scar tissue was visible in the occluded animals, and gene expression changes in the infarct and borderzone were in agreement with previous studies of MI. Library preparation was completed using the TruSeq Stranded Total RNA kit (Illumina), and sequencing using the Illumina HiSeq 2500 Sequencer. Sequence alignment and QC was performed using STAR. RNA expression across all samples was highly consistent, however small yet consistent changes in the expression of genes were detected between mice with MI and controls. These changes included genes encoding ion channels, receptors, synaptic proteins and actin remodeling proteins.

Support or Funding Information

This work was supported by NIH RO1 HL093056
Comparison Between Spectral Analysis and Symbolic Dynamics for Heart Rate Variability Analysis in the Rat
Authors: Silva Luiz Eduardo Virgilio , Geraldini Victor Rezende , Oliveira Bianca Potratz , Silva Carlos Alberto Aguiar , Salgado Helio Cesar , Fazan Rubens
Published: November 2023

Although spectral analysis has been widely used to assess the autonomic control of the heart, several issues are still in debate about its interpretation, especially in experimental animals such as rats and mice. A nonlinear alternative method, namely symbolic dynamics, was proposed and has been reported to be very useful to represent sympathetic and parasympathetic modulation to the heart. However, studies evaluating the validity of symbolic dynamics in experimental models still lack. Conscious male Wistar rats, under continuous recording of electrocardiogram (ECG) or arterial pressure (AP), were subjected to autonomic imbalance by two protocols: pharmacological blockade of adrenergic or cholinergic muscarinic receptors administration (atenolol 4 mg/kg or methylatropine 2 mg/kg, iv, N=9 in each group) and reflex changes in sympatho‐vagal balance by AP changes elicited by phenylephrine (PHE, N=9) or sodium nitroprusside (NPS, N=9). Beat by beat series of cardiac interval (CI) were generated from ECG or AP recordings and both spectral analysis and symbolic dynamics were calculated for each animal. Spectra of CI series were calculated by FFT and integrated in low‐ (LF: 0.2–0.8Hz) and high‐frequency bands (HF: 0.8–3Hz). For symbolic analysis, symbols were attributed to CI values and sequences of 3 symbols were classified as 0, 1 or 2V according to the number of variations showed. Percentage of 0V and 2V are associated to sympathetic and vagal cardiac modulation, respectively. Hypertensive response caused by PHE (33 ± 3 mmHg) led to a reflex bradycardia (−45 ± 3 bpm) while hypotension due to NPS (−15 ± 1 mmHg) was accompanied by tachycardia (46 ± 6 bpm). The administration of atenolol and atropine led to a reduction and an increase in HR, respectively (−19 ± 6 and 70 ± 8 bpm). All drugs used reduced the total variability (standard deviation) of the CI series. The results of the spectral and symbolic analysis are showed in the table at the end. The symbolic dynamics proved to be more robust than the spectral analysis for the evaluation of the autonomic control in rats. The 1V family patterns, hitherto unknown, have been shown to be linked to sympathetic modulation in the rat and should be considered in future studies.

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Fapesp

basal
atenolol
basal
atropine
basal
NPS
basal
PHE

LF (nu)
35±2
23±4*
40±4
28±4*
9.8±2.1
14±4
11±2
9.3±2.0

HF (ms2)
6.0±0.8
2.7±0.9*
4.3±0.4
0.2±0.03*
10±2
6.3±1.5*
4.2±1.0
25±6*

0V (%)
27±2
16±3*
27±3
29±4
7.7±1.4
9.1±1.7
9.0±1.4
5.6±1.2

1V (%)
44±1
38±2*
42±1
46±2
35±2
40±1*
45±2
33±2*

2UV (%)
25±2
37±4*
26±2
19±1*
50±3
43±2*
39±3
51±4*

P<0.05 compared to basal.
The Effect of a Patent Foramen Ovale on Ventilatory Responses to Hypoxia
Authors: Chang Alex J , Boulet Lindsey M , Davis James T , Hardin Alyssa M , Foster Glen E , Lovering Andrew T
Published: November 2023

Approximately 35% of the general healthy population has a patent foramen ovale (PFO) (Woods et al., 2010; Marriott et al., 2013; Elliott et al., 2013). Previous work by our lab has shown that after 16 days of exposure to 5260 m, subjects with a PFO (PFO+) had blunted ventilatory acclimatization to high altitude compared to subjects without a PFO (PFO−), such that PFO+ subjects had a lower PaO2, higher PaCO2, and lower SaO2 (Elliott et al., J Appl Physiol, 2015). However, in that study 7/11 PFO+ subjects were female whereas only 2/10 PFO− subjects were female and thus potential sex differences were not accounted for and it is known that sex hormones can affect ventilatory responses to O2 and CO2 (Schoene et al., 1986; Slatkovska et al., 2006). Thus, it remains unknown if PFO+ subjects exhibit blunted ventilatory responses to hypoxia compared to PFO− subjects, independent of sex. Therefore, the purpose of this study was to determine if the presence of a PFO affects ventilatory responses during acute exposure to either poikilocapnic hypoxia or isocapnic hypoxia.

A total of 31 healthy, non‐smoking subjects matched for height, weight, sex and age completed the entire study:

PFO+: age: 24 ± 5 yrs, height: 170 ± 11 cm, weight: 69 ± 14 kg, BSA: 1.8 ± 0.2 m2

PFO−: age: 27 ± 9 yrs, height: 172 ± 10 cm, weight: 71 ± 16 kg, BSA: 1.8 ± 0.2 m2

These 31 subjects included 15 PFO+ subjects (8 female) and 16 PFO – subjects (8 female). On the day of the test, the subjects came to the lab on a single visit and breathed 1) poikilocapnic hypoxia (PETO2 = 51.4 ± 6.5 mmHg) and 2) isocapnic hypoxia (PETO2 = 43.7 ± 4.4 mmHg, PETCO2 = 40.0 ± 3.4 mmHg), and these gases were administered using the AirForce breathing response system in a randomized and balanced order. Time points of interest were baseline resting ventilation while sitting comfortably in a chair and the time at which VE was greatest after 5 minutes of breathing the respective hypoxic gas. There was a 40 min break between hypoxia trials. Hypoxic ventilatory response (HVR) was calculated as the change in minute ventilation divided by the change in saturation (Δ VE/Δ SpO2).

There was no effect of PFO on HVR during exposure to either isocapnic hypoxia or poikilocapnic hypoxia (p > .05). There was no effect of PFO on SpO2, VE, VT, RR, PETO2 or PETCO2 during either the isocapnic hypoxia or poikilocapnic hypoxia trials (p > .05). These data suggest that HVR is not blunted in PFO+ subjects compared to PFO− subjects. Additionally, there were no sex differences in HVR in PFO+ subjects compared to PFO− subjects. Based on these findings, it is plausible that the blunted ventilatory acclimatization previously found by our group may be due in part to differences in responses to hypercapnia suggesting involvement of a central chemoreceptor rather than just a peripheral component (see accompanying poster Hardin et al 2016).
Ventilatory, metabolic and thermal responses to hypercapnia and hypoxia in orchidectomized and testosterone replaced adult male Wistar rats
Authors: Marques Danuzia A , de Carvalho Debora , da Silva Glauber SF , Scarpellini Carolina da S , Bícego Kênia C , Szawka Raphael E , Gargaglioni Luciane H
Published: November 2023

Elevated plasmatic concentrations of Testosterone (T) may have an impact over respiratory parameters. Therefore, we evaluated the influence of T on ventilation (VE) and oxygen consumption (VO2) during room air, hypercapnia (7%CO2) and hypoxia (7%O2) in adult male Wistar rats. VE measurements were obtained using the barometric method (whole body plethysmography) and VO2 by closed respirometry system in intact and orchidectomized animals treated with vehicle (corn oil, ORX group; 0.2 mL/rat, s.c.) or T propionate (ORX+T group; 0.25 mg/0.2 mL/rat, s.c) for seven days. Orchiectomy did not affect ventilatory, metabolic and thermal responses to hypercapnia and hypoxia in rats. T replacement caused an increase in ventilatory response to hypercapnia (VE/VO2‐intact: 87.2 ± 9.8; ORX: 78.9 ± 13.3 and ORX+T: 124.4 ± 21.7) and to hypoxia (VE/VO2‐intact: 96.7 ± 17.5; ORX: 85.1 ± 16.1 and ORX+T: 119.5 ± 18.2). Our data suggest that T exerts an excitatory modulation in central and peripheral chemoreflex.

Support or Funding Information

FAPESP and CNPq
Dynamic characteristics of respiratory central chemoreflex in normal rats
Authors: Tohyama Takeshi , Saku Keita , Tsutsui Hiroyuki , Sunagawa Kenji
Published: November 2023

Background

Central chemoreflex is the dominant regulator of respiration. Central chemoreceptors are located on the ventral surface of the medulla, respond primarily to changes in arterial carbon dioxide content (PaCO2). It is well known that increasing in PaCO2 linearly increases minute ventilation (VE) and increasing in VE decreases PaCO2 with hyperbolic manner. Thus, central chemoreflex consists of a negative feedback loop to maintain PaCO2 around 40 mmHg. Although the static characteristics of central chemoreflex have been well examined in both animals and human, the dynamic characteristics, which is prerequisite to understand how quickly and how stably central chemoreflex operates to regulate ventilation and PaCO2, has not been elucidated. The purpose of this investigation is to characterize the dynamic characteristics of central chemoreflex control of ventilation and PaCO2 with a system identification techniques based on a white noise approach.

Methods

We divided the central chemoreflex system into two components: central controller (EtCO2‐VE relationship) and peripheral plant (VE‐EtCO2 relationship) (Fig. 1). In eleven anesthetized rats, we measured respiratory end tidal CO2 (EtCO2) as a surrogate of PaCO2, and VE. To estimate central controller, we randomly switched inhaled gas from 0% to 5% CO2 every 10 sec for an hour and recorded spontaneous VE (Fig. 2A). To evaluate the peripheral plant, we randomly switched VE from hypoventilation to hyperventilation every 0.8 sec for an hour and simultaneously recorded EtCO2 under mechanical respiratory condition (Fig. 2B). We calculated the transfer functions of central controller and peripheral plant in the frequency domain.

Results

The gain of central controller was flat up to 0.005 Hz and then decreased with frequencies. The phase was in phase at the lowest frequency and gradually delayed in higher frequencies (Fig. 3A). In the peripheral plant, the gain gradually decreased from the lowest frequency to 0.1Hz and then sharply decreased. Its phase has out of phase at the lowest frequency and gradually delayed in higher frequencies (Fig. 3B). Transfer functions of both subsystems represented as a low‐pass filter characteristic with dead time. High coherence values suggested that the input‐output relationships of the central controller and peripheral plant were predominately governed by this framework.

Conclusions

We characterized the dynamic properties of the central controller and peripheral plant of the central chemoreflex in normal rats with the white noise system identification method. These systematic analysis of chemoreflex dynamics would help us understand the respiratory regulation in physiological condition.