bluuebunny/crossref_metadata_2025 huggingface dataset:
Reveal the Role of Store‐Operated Calcium Entry in Epirubicin‐induced Acute Oxidative Stress in Cardiomyocytes
Authors: Liu Xian , Pan Zui
Published: June 2021

Epirubicin (EPI) is a widely used anthracycline chemotherapy drug, whose use is limited by its side effects including cardiomyopathy, hypertrophy and chronic heart failure. The cardiotoxicity of EPI has been linked to the generation of reactive oxygen species (ROS) in mitochondria and disruption of cardiac ryanodine receptor Ca2+ release channel in the sarcoplasmic reticulum membrane. As Store‐Operated Calcium Entry (SOCE) is important in regulating intracellular Ca2+ signaling, we tested whether dysregulated SOCE was also involved in EPI‐induced acute cardiotoxicity. This study compared intracellular ROS levels using fluorescent probe dihydroethidium (DHE) in EPI‐treated HL‐1, an adult mouse atrial cardiomyocyte cell line. Treatment of EPI at 1uM for 30min significantly increased intracellular ROS levels in HL‐1 compared with vehicle‐treated cells (3.8fold, p<0.0001). HL‐1 cells were then treated with 20uM BTP2, a potent SOCE inhibitor, for 30min. However, BTP2 failed to significantly decrease the EPI‐induced intracellular ROS (p=0.9987, n=8 replicates). In addition, 50uM ethylene glycol‐bis (β‐aminoethyl ether)‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (EGTA‐AM) failed to prevent EPI‐induced ROS increase (p=0.9768, n=8 replicates). These results might be limited by the technical challenge such as DHE not able to quantitatively distinguish intracellular ROS levels and EGTA‐AM might not be fast enough to chelate Ca2+ from Ca2+ influx. Ongoing project will seek to overcome the technical limitation to further test the role of SOCE in EPI‐induced acute cardiotoxicity.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of a new Dent Disease mouse model carrying a pathogenic mutation of ClC‐5
Authors: Sakhi Imene , Bignon Yohan , Frachon Nadia , Lourdel Stephane
Published: June 2021

Dent Disease is a rare X‐linked inherited renal proximal disorder characterized by low molecular weight (LMW) proteinuria, hypercalciuria, kidney stones and progressive renal failure. Inactivating mutations of the CLCN5 gene encoding the 2Cl−/H+ exchanger ClC‐5 have been identified in approximately two‐thirds of patients with Dent Disease. ClC‐5 is predominantly expressed in early endosomes of proximal tubules where it optimizes the function of a vacuolar H+ pump to ensure an efficient endocytosis of LMW proteins.

We have previously investigated the functional consequences of CLCN5 mutations in heterologous expression systems and shown that 60 % of the mutations lead to a defect in protein folding and processing. As a consequence, the misfolded ClC‐5 are retained within the endoplasmic reticulum (ER) where they could induce ER stress response. Such ER stress might in turn induce oxidative stress in proximal tubule cells. To investigate the consequences of intracellular retention of a misfolded ClC‐5, we created a mouse model carrying the N340K pathogenic mutation.

ClC‐5 expression and localization in WT and N340K male mice were studied by Western blot and on kidney sections by immunofluorescence. Daily food, water intake, body weight and urine excretion data were collected in metabolic cages for N340K and WT mice. Urine as well as blood samples were analysed for ions, protein, glucose and creatinine to detect kidney dysfunction as observed in patients with Dent Disease.

Mice carrying the N340K mutation displayed ClC‐5 intracellular retention in proximal tubules cells. They showed an increased urinary calcium and glucose excretion and a decreased urinary pH. They also had a severe LMW proteinuria, recapitulating common features of Dent Disease.

The present study validates a new Dent Disease mouse model carrying a pathogenic Clcn5 mutation retained in the ER. The potential link between ClC‐5 retention in the ER, ER stress and oxidative stress responses has yet to be demonstrated.

Support or Funding Information

Prix “Jeune Chercheur”, Fondation du Rein 2017

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological Inhibition of Glucose‐6‐phosphate Dehydrogenase Produces Inhibition of L‐type Calcium Channel Current in Arterial Smooth Muscle Cells
Authors: Ochi Rikuo A. , Chettimada Sukrutha , Kizub Igor , Gupte Sachin A.
Published: June 2021

Glucose‐6‐phosphate dehydrogenase (G6PD) is the first enzyme in pentose phosphate pathway (PPP). G6PD is involved in cellular redox regulation by reducing NADP+ to NADPH, a primary cellular reducing agent, and PPP produces ribose 5‐phosphate, a substrate necessary for nucleotide synthesis. We used two inhibitors of G6PD, 6‐aminonicotinamide (6‐AN) and dehydroepiandrosterone (DHEA), to clarify involvement of G6PD in the regulation of L‐type Ca2+ channel current (ICa,L) in arterial smooth muscle cells (ASMCs). 6‐AN is converted to 6‐ANADP in the cell to competitively inhibit NADP+ binding to G6PD. DHEA is an adrenal steroid hormone with the highest serum concentration among steroid hormones with its sulfate ester DHEAS. DHEA inhibits G6PD in a non‐competitive manner. The potency of DHEAS to inhibit G6PD is about 1/100 of DHEA. DHEA induced relaxation of high K+‐induced contraction in arterial strips excised from carotid artery and aorta of rat in a dose‐dependent manner. In contrast, DHEAS did not produce the relaxation. In previous meetings we reported that DHEA inhibits ICa,L in voltage‐dependent and ‐independent mechanisms in ASMCs. 6‐AN produced voltage‐independent inhibition of ICa,L recorded with 10 mM Ba2+ as the charge carrier in A7r5 ASMCs. When constant depolarization step was repetitively applied from hyperpolarized HP, 6‐AN (3 mM) gradually induced inhibition of ICa,L decreasing the peak amplitude of ICa,L (ICa,L, peak) by 25% in 5 min and 45% in 10 min. The inhibition was not reversible by washout of 6‐AN. This was in contrast to DHEA‐induced inhibition of ICa,L which exhibits rapid onset and rapid recovery by washout. 6‐AN little affected the time course of inactivation of ICa,L during depolarization steps, while DHEA markedly accelerated the time course. When I‐V relationship was obtained with 5 min interval before and during application of 6‐AN, ICa,L did not significantly change in control, but decreased in 5 min in the presence of 6‐AN and the inhibition was increased by another 5 min application of 6‐AN. In the presence of 6‐AN, 0.1 mM DHEA rapidly decreased ICa,L,peak and markedly accelerated the time course of inactivation in a reversible manner. Inhibition of G6PD by 6‐AN and DHEA could inhibit ICa,L,peak by thiol‐oxidation of CaV1.2 channel proteins via decrease of NADPH. DHEA additionally augmented voltage‐dependent inactivation of ICa,L in a mode of open channel block produced by Ca2+ channel blockers.

Support or Funding Information

National Heart, Lung, and Blood Institute Grant RO1‐HL‐085352 and ROI‐HL‐132574 to S.A.G.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endothelial Upregulation of Mechanosensitive Channel Piezo1 in Pulmonary Hypertension
Authors: Wang Ziyi , Babicheva Alexandra , Wu Linda , Yuan Jason X.‐J. , Wang Jian
Published: June 2021

Rationale

The Piezo channel family (Piezo1 and Piezo2), one of the most important family of mechanosensing ion channels. Surprisingly, there are no studies on Piezo in the pulmonary hypertension. We focus on Piezo1 because of its imprtance in vascular deveopment, especially in endothelial cells. Our working hypothesis is that upregulated Piezo1 may be a critical pathogenic mechanism that contributes to augmented Ca2+ influx and excessive PAEC proliferation or apoptosis in patients with IPAH.

Methods

RNA levels of Piezo1 and Piezo2 was quantified by RT‐PCR Analysis. Protein levels of Piezo1, bax, bcl‐2, Calpain‐1 and Calpain‐2 was quantified by Western blot analysis. adult Male C57BL/6 mice (6–8 weeks) were exposed to persistent chronic hypoxia (10% O2) for 21 days to establish hypoxia‐induced PH. For Sugen 5416/hypoxia (SuHx) induced‐PH rat model, SD rats (age, 6–8 weeks old) were either injected s.c. with 20 mg·kg−1 SU5416 (Sigma‐Aldrich, St. Louis, MO, USA) or vehicle, exposed to hypoxic conditions for 3 weeks (10% O2) and returned to normoxic condition (21% O2) for 2 weeks. In the MCT group, a single intraperitoneal injection of MCT (60 mg/kg, Sigma Aldrich) was given at day 1 to establish the MCT‐induced PH.

Results

Piezo1 and Piezo2 have high expression in rat lung tissue and human PAEC, and also Piezo1 has significantly higher mRNA levels in both human PASMC and PAEC than Piezo2 (n=5, p<0.01). Upregulated Piezo1 in whole‐lung tissue (mainly containing ECs) from MCT‐mediated PH (MCT‐PH) compared to normal control rats (1.00±0.07; 4.74±0.41, n>5, p<0.001). Compared to controls, Piezo1 was increased in whole‐lung tissues in hypoxic mice (1.00±0.07; 4.74±0.41, n>5, p<0.01) and SuHx rats (1.00±0.15; 1.99±0.31, n>5, p<0.01). Also, compared to normoxia condition, increased Piezo1 was associated with a increased Calpain‐1 and Calpain‐2 treated with hypoxia (3% O2 for 72 hours) (1.00±0.16; 4.14±0.75; 1.00±0.14, 2.32±0.35; 1.00±0.07; 1.74±0.14, n>5, p<0.01). Moreover, PAECs from IPAH patients exhibited significant elevation of Piezo1 compared to normal control subjects (1.00±0.09; 6.20±1.78, n=5, p<0.01).

Conclusions

These results are the first to implicate that Piezo1 as a mechanosensor may play an important role in the development of pulmonary hypertension via, at least in part, induction of PAEC apoptosis and enhancement of Ca2+ in PAECs. Piezo1 may be involved in the transcriptional regulation under hypoxia and play critical roles in the development of PH. Therefore, further studies are needed to identify the potential contribution of Piezo1 in PH.

Support or Funding Information

None.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vaping During Pregnancy Results in Arterial Stiffness in Offspring
Authors: Aboaziza Eiman , O'Reilly Juliana , Moore Josh , McLaughlin Sarah , Olfert I. Mark , Chantler Paul
Published: June 2021

Electronic cigarettes (Ecigs) are promoted as effective smoking cessation tools, and consequently widely used among pregnant women who are motivated to quit tobacco cigarette smoking. Emerging evidence demonstrates that Ecigs can be dangerous to cardiovascular health. Babies born to women who vape represent a uniquely vulnerable population for this type of exposure, because they may be adversely impacted during a critical developmental period. It is unknown if cardiovascular harm potential of Ecigs without nicotine is conferred to offspring of vaping mothers. Pulse wave velocity (PWV) provides an indirect measure of arterial stiffness (with stiffer vessels having higher PWV) and can be useful for characterizing cardiovascular disease progression.

Sprague‐Dawley rat dams were exposed to either nicotine‐free Ecig vapor (Ecig0, Joyetech eGrip OLED using 5‐sec puffs @17.5 W) or ambient air. Exposure consisted of 20 puffs over 1‐hour each day, 5 days/week, and resulted in an average daily TPM of ~120 mg/m3. Maternal exposure started on gestational day 2 and continued until pups were weaned (postnatal day 21). Pups were never directly exposed. Left common carotid arteries (LCCA) of 4‐month old pups (n=5 per group) were noninvasively imaged via Doppler ultrasound. PWV was measured using the regional transit‐time (TT) method (PWV=Δd/Δt; where d is distance between proximal (downstream to aortic arch) and distal (upstream of bifurcation) points on the LCCA, and t is the arrival time of pulse wave upstroke relative to R‐wave peak on ECG). Repeated (≥3) measurements were made for each variable and averaged. TT measurements were performed using a Vevo2100 ultra high frequency ultrasound platform with heart rate monitoring system (VisualSonics Inc, Toronto, ON, Canada). Results were processed using VisualSonics analysis software.

Left common carotid arterial PWV (mean ± SD) in the Ecig0 group (6.6 ± 2 m/s) was significantly higher (p<0.01) than in the air control group (3.2 ± 0.7 m/s).

Offspring exposed to nicotine‐free Ecig vapor in utero had higher PWV than controls, indicating stiffer arteries and an increased risk for cardiovascular disease. Further studies will determine the long‐term implications of this vascular impairment. Clinicians and policy‐makers need to be aware of these risks to discourage pregnant women from using Ecigs (with or without nicotine).

Support or Funding Information

WVU Cancer Institute Philip R Dino Innovative Research Grant (IMO); APS STRIDE Fellowship (JO); AMIF: NIH S10RR026378, P20RR016440, P30GM103488, P20RR016440, P30GM103488 and U54GM104942 (SM)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Signaling of IL13 through IL4Ra Promotes Cardiac Regeneration
Authors: Paddock Samantha J , Wodsedalek Dylan J , Kenarsary Aria , O'Meara Caitlin C
Published: June 2021

Introduction

During the first week of life, neonatal mice are able to regenerate their hearts after injury with minimal scarring. Our lab has previously published that the global knockout of anti‐inflammatory cytokine Interleukin 13 (IL13) inhibits neonatal cardiac regeneration in mice. IL13 primarily acts through the IL4Ra/IL13Ra1 heterodimer receptor to promote cell cycle activity. However, how IL13 specifically signals in the heart to promote this phenotype remains unexplored. We hypothesize that the deletion of the IL4Ra component of IL13's receptor complex will lead to a similar lack of regeneration in the mouse heart.

Methods

To delineate IL13 signaling mechanisms in the mouse, we utilized various knockouts of IL13 signaling—IL13 global knockout mice, IL4Ra global knockout mice, and C57/BL6 wildtype mice. With cardiomyocytes (CMs) isolated from these models, we analyzed the dynamics of downstream factors of recombinant IL13 (rIL13) in vitro. Additionally, at postnatal day 1 (P1), our mouse models were given apical resections to remove up to 10% of the apex of the heart. After resection, we investigated mRNA and protein expression of downstream markers, as well as CM proliferation at different time points. We also analyzed overall regenerative potential through echocardiography and Gömöri trichrome staining.

Results

In vitro administration of rIL13 to wildtype neonatal mouse CMs resulted in strong, but transient activation of AKT and ERK1/2, which have been shown to be pro‐survival and pro‐proliferative respectively. In our IL4Ra KO mice, we see an attenuation of this signaling. At 7 days post resection (dpr), we see a significant decrease in proliferating CMs from IL13 KO mice when compared to wildtype. Additionally, at 21dpr in vivo, we observe significant fibrotic apical scars in IL13 KO mice, indicating lack of cardiac regeneration.

Conclusion

From these results, our data suggests that depletion of IL13 and its receptor, IL4Ra, inhibits regenerative pathways in the heart.

Support or Funding Information

American Heart Association

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Novel Role of Thbs‐1/CD47‐Drp1 signaling following ischemia reperfusion in the aging heart
Authors: Kelm Natia , Beare Jason , Yuan Fangping , LeBlanc Amanda
Published: June 2021

Ischemia reperfusion (IR) injury leads to activation of dynamin‐related protein (Drp‐1), causing mitochondrial fission and generation of reactive oxygen species (ROS), but the molecular mechanisms that activate Drp‐1 are not known. The purpose of this study was to establish the signaling pathway between Thbs‐1/CD47 and fission protein (Drp‐1) through PGC‐1 following IR in advancing age.

Methods

Female Fischer‐344 rats were divided into 4 groups: Young Control, Young+IR, Old Control and Old+IR. Echocardiography measurements were done at baseline and 72 hours after IR (30 minutes of ischemia,72 hrs of reperfusion). After evaluation of heart function and coronary flow using vevo3100, hearts were explanted and mitochondrial ROS generation was measured using MitoPY1 (O2·−), as well as protein levels of Thbs‐1, PGC‐1, and Drp‐1. In vitro, rat aortic endothelial cells (RAEC) received hypoxic conditions to mimic IR in vivo model. Cells were treated with SiRNA for PGC‐1 or control scrambled SIRNA and then underwent hypoxia challenge to evaluate PGC‐1 effect on Drp‐1. Cell lysate was collected for Western Blot after 36 hours of transfection.

Results

Mitochondrial O2·− generation in heart tissue increased in both age groups following IR. Old animals experiencing diastolic dysfunction at the baseline, after IR they exhibited reduced systolic function and exacerbated diastolic dysfunction compared to young controls. IR induces Thbs‐1 and Drp‐1 expression in young and old hearts. SiRNA to PGC‐1 enhanced levels of Drp‐1 in RAECs and increases ROS generation after hypoxia.

Conclusion

Left ventricular diastolic dysfunction during IR is potentiated due to increased generation of ROS. These results highlight a novel signaling pathway by which Thbs‐1 regulates mitochondrial fission (Drp‐1) and ROS generation during hypoxia, and presumably, following IR. Inhibiting Thbs‐1 immediately after IR may prevent Drp‐1‐mediated mitochondrial fission and is likely to improve diastolic function of the heart by reducing ROS‐mediated cardiomyocyte damage in the aged population.

Support or Funding Information

NIH R01 AG05358 Gheen's Foundation

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Vascular Contributions to Intracranial Hemodynamic Pulsatility and Pulsatile Dampening in Adults
Authors: Lefferts Wesley K , DeBlois Jacob P , Augustine Jacqueline A , Heffernan Kevin S
Published: June 2021

The brain is a high‐flow organ that requires continuous, non‐pulsatile blood flow. Intracranial blood flow pulsatility depends on the ability of the large elastic arteries and cerebrovasculature to dampen pulsatile hemodynamics transmitted from the heart. Aging is accompanied by large artery stiffening and reductions in cerebrovascular dampening, ultimately increasing intracranial pulsatility. It is unclear what hemodynamic forces may be responsible for these age‐related changes in the transmission and dampening of intracranial pulsatility across the lifespan. The purpose of this study was to explore hemodynamic contributors to intracranial pulsatility and pulsatile dampening in 244 adults between 35–85 yr (49% female, 56±12 yr, 27±4.2 kg/m2). Intracranial pulsatile dampening was calculated as the ratio between common carotid and middle cerebral artery (MCA) blood velocity pulsatility index (PI) measured via Doppler. Carotid ultrasound and wave intensity analysis were used to measure carotid diameter, forward compression wave energy (W1), wave reflection index (RIx), and forward traveling expansion wave (W2). Aortic (carotid‐femoral) and carotid pulse wave velocity (PWV) were assessed via tonometry and carotid ultrasound, respectively, and expressed as a ratio (aortic:carotid PWV). Carotid pulse pressure (PP) was measured via tonometry. Multiple regression was used to examine the contributions of 1) age, sex, BMI, aortic:carotid PWV, carotid diameter, PP, and W1 to intracranial pulsatility (MCA PI); and 2) age, sex, BMI, aortic:carotid PWV, carotid diameter, RIx, and W2 to intracranial pulsatile dampening. After entering all variables, significant predictors of MCA PI included age (β,0.44), aortic:carotid PWV (β,0.16), PP (β,0.29), and W1 (β,0.20; R2=0.50, p<0.01). Significant predictors of intracranial pulsatile dampening included age (β,−0.47), sex (β,−0.43), and RIx (β,0.26; R2=0.53, p<0.01). Our data confirms recent literature linking age, exaggerated stiffening of the aorta compared to carotid artery, and extracranial hemodynamics to intracranial pulsatility in the MCA. Moreover, our findings indicate age, sex, and wave reflections in the carotid artery impact the ability of the cerebrovasculature to dampen transmission of pulsatile hemodynamics. These data highlight the potential utility of the large extracranial arteries as therapeutic targets to reduce intracranial pulsatility and importance of carotid wave reflections in dampening intracranial pulsatility.

Support or Funding Information

Funded in part by an ACSM Foundation Research Grant (WKL), AHA Predoctoral Fellowship (WKL), and Dairy Research Institute/Dairy management Inc. Grant 1154 (KSH).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Reduction in Cardiac Function Precedes Structural Adaptations in Experimental Spinal Cord Injury
Authors: Fossey Mary , Poormasjedi‐Meibod Malihe‐Sadat , Hayes Brian , Erskine Erin Laurel , Azad Reathm K , Granville David James , Ramer Matt Steven , West Christopher Roy
Published: June 2021

Introduction

Chronic high‐thoracic spinal cord injury (SCI) is associated with systolic dysfunction, cardiomyocyte atrophy and the upregulation of major proteolytic pathways in the heart. How such dysfunction manifests with time is presently unknown.

Methods

Male Wistar rats underwent complete SCI at the 3rd thoracic spinal level (T3‐SCI; n=10) or dorsal durotomy (SHAM; n=5). T3‐SCI rats were terminated at different time points: three days post‐SCI (3‐day SCI; n=5) or seven days post‐SCI (7‐day SCI; n=5). SHAM rats were terminated at seven days post‐durotomy. On termination day, prior to euthanasia, left‐ventricular (LV) catheterization was performed to assess cardiac function. Additionally, cardiac tissue was collected for histological analysis to quantify cardiomyocyte dimensions (standardized to femur length).

Results

Relative to SHAM, T3‐SCI rats at both 3‐day and 7‐day post‐SCI exhibited reduced stroke work (SW; p=0.036), load‐independent contractile function (inferred with end‐systolic pressure‐volume relationship, ESPVR; p=0.0013) and maximal rate of LV systolic pressure increment (dP/dtmax; p=0.0070). The maximal rate of LV relaxation (i.e. ‐dP/dtmin; p=0.019) was reduced at 3‐day and 7‐day post‐SCI vs. SHAM; however, tau (time constant of isovolumic relaxation) was not different (p=0.15). There were no differences for any functional indices between 3‐day vs. 7‐day SCI. Histological analysis of cardiomyocytes indicates no significant differences in length and width in SCI groups vs. SHAM (length p=0.20; width p=0.24).

Conclusion

Systolic cardiac function decreased significantly in both 3‐day and 7‐day SCI rats vs. SHAM. This indicates a rapid onset of cardiac dysfunction following T3‐SCI. Cardiomyocyte atrophy was not present in either 3‐day or 7‐day SCI rats, implying a reduction in cardiac function precedes structural remodeling.

Support or Funding Information

Heart and Stroke Foundation of Canada (HSFC), International Collaboration on Repair Discoveries (ICORD) and Blusson Integrated Cure Partnership (BICP).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dislocation of Lysyl Oxidase and Deformation of Cardiomyocytes in Response to Myocardial Ischemic Injury in Mice
Authors: Chu Qing , Xiao Ying , Song Xin , Kang Y. James
Published: June 2021

Lysyl oxidase (LOX), a copper‐dependent amine oxidase, is located in the intercalated discs between cardiomyocytes in the heart and responsible for the catalysis of collagen and elastin cross‐linking of the extracellular matrixes. However, in response to myocardial ischemic injury, LOX is dislocated to the cytosol, along with deformation, of cardiomyocytes in the ischemic myocardium. The present study was undertaken to explore the relationship between LOX dislocation and the deformation of cardiomyocytes in response to myocardial ischemia. Male C57BL/6J mice aged 8–12 weeks old were subjected to left anterior descending (LAD) coronary artery ligation to induce ischemic injury. The hearts were collected at 1st, 4th, or 7th day after the surgery and cut into frozen/paraffin sections to detect LOX location and the morphological changes of cardiomyocytes. Most of the survived cardiomyocytes in the ischemic infarct area were deformed from the rod‐shaped to the rounded detected on the 7th day after the surgery. Corresponding to the deformation of cardiomyocytes, the junction between cardiomyocytes become disturbed, and the intercalated discs disappeared. Connexin 43 (Cx43), an important gap junction protein located in the intercalated discs, was significantly reduced in the ischemic infarct area at day 1, but slightly recovered at day 7 after LAD ligation. However, the recovered Cx43 proteins were mainly located in the cytosol of cardiomyocytes. LOX proteins were increased gradually from day 1 to day 7 in the ischemic infarct area in comparison with the sham‐operated controls, and were mostly dislocated in the cytosol of cardiomyocytes. The results suggest that in response to ischemic injury, dislocation of LOX in cytosol of cardiomyocytes is likely responsible at least in part for the breakage of the junction between cardiomyocytes, leading to loss of the cell polarity and impaired contractile function of cardiomyocytes.

Support or Funding Information

Scientific research funds of West China Hospital, Sichuan University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
PP242, mTOR inhibitor, decreases phenylephrine‐induced vascular contractility in hypertensive and normotensive arteries
Authors: Quinones Patricia Martinez , Patel Viral , Lewis Steven , McKenzie Jaine , Warner Alex , McCarthy Cameron , Wenceslau Camilla , Komic Amel , Webb R Clinton
Published: June 2021

Cardiovascular disease is the leading cause of death in cancer survivors. The short‐term and long‐term cardiotoxic effects of targeted chemotherapeutics is not well known. PP242, a dual inhibitor to mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and 2 (mTORC2), is a targeted therapy in clinical trials for various malignancies including bladder, ovarian and gastric cancer. mTORC1 inhibition has been associated with heart failure, while mTORC2 inhibition has been considered cardioprotective. We hypothesized that PP242 leads to enhancedcontractile response to phenylephrine (PE), and impaired Acetylcholine (ACh)‐induced relaxation. We studied the effects of PP242 on the contractility and relaxation of aortic rings and mesenteric resistance arteries (MRA) of male Wistar rats (12–15 weeks old, n=4) and spontaneously hypertensive rats (SHR) (15–18 weeks old, n=6) on a wire myograph. Vessel segments were exposed to PP242 (500nM) for one‐hour incubation. Normotensive aortic rings exposed to PP242 showed a decreased contractile response to PE (LogEC50±SEM, Ctrl:−7.617±0.178 vs Drug: −7.292±0.129) and increased relaxation to ACh (LogEC50±SEM, Ctrl:−6.917±0.164 vs Drug: −7.588±0.164). SHR aortic rings exposed to PP242 (LogEC50±SEM, Ctrl:−7.606±0.129 vs Drug: −7.124±0.118) showed decreased contractility but no difference in ACh‐induced relaxation. No differences were observed in normotensive MRA. SHR MRA showed decreased PE‐induced contraction (LogEC50±SEM, Ctrl:−5.402±0.060 vs Drug:‐−5.322±0.065) and increased ACh‐induced relaxation (LogEC50±SEM, Ctrl:−7.992±0.117 vs Drug:−8.139±0.092). Our data suggest that dual inhibition of mTORC1 and mTORC2 with PP242 may provide a cardioprotective effect by limiting vasoconstriction and may be a good therapeutic agent for hypertensive cancer patients. However, long term treatments with this targeted therapy need to be evaluated for vascular dysfunction induction, as the hypocontractile effect could contribute to failure of the cardiovascular system, which could be associated with cardiotoxicity.

Support or Funding Information

NIH P01 HL134604 (RCW), k99GM11888 (CFW)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Therapeutic Telomerase Reactivation for the Prevention of Doxorubicin‐Induced Cardiotoxicity
Authors: Chatterjee Shambhabi , Hofer Teresa , Gupta Shashi Kumar , Thum Thomas , Bär Christian
Published: June 2021

Background

Doxorubicin (DOX) is one of the most potent chemotherapy agents. Its clinical use however, is restricted due to the severe risk of cardiotoxicity, presumably attributed to high levels of reactive oxygen species (ROS) leading to cardiomyocyte death. Telomerase prevents cell death through maintaining telomeres. In addition, telomerase has non‐canonical, anti‐apoptotic and pro‐survival traits, likely owing to detoxification of ROS. However, telomerase is silenced in adult murine and human hearts.

Purpose

To test whether pharmacological reactivation of telomerase may be effective as a therapeutic strategy to prevent doxorubicin‐induced cardiotoxicity.

Methods and Results

We used a AAV9‐based gene therapy approach to reinstate telomerase expression in cardiomyocytes in a mouse model of chronic DOX‐induced cardiotoxicity. Telomerase treated mice presented with preserved heart weights in comparison to AAV‐empty controls after DOX treatment. Histopathological analyses revealed that this rescue was accompanied by preserved cardiomyocyte size and reduced apoptosis rates. This was also reflected by improved cardiac function parameters determined by echocardiography and Millar catheterization. At the molecular level we observed altered mitochondria morphology and reduced mitochondrial fission in response to telomerase expression. In line, Western blotting post telomerase reactivation revealed lower levels of the mitochondrial fission protein Drp1, which plays a known role in cardioprotection. Complementary in vitro experiments confirmed anti‐apoptotic effects of telomerase in DOX treated human induced pluripotent stem cell (hiPSC)‐derived cardiomyocytes.

Conclusion

Our data suggest that cardiac‐specific telomerase reactivation could be a novel strategy for the prevention of doxorubicin‐induced cardiotoxicity in cancer survivors.

Support or Funding Information

This work was supported by the DFG (BA5631/2‐1 to CB) and ERA‐CVD Network (EXPERT grant to TT).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Cardioprotective and Hypocholesterolemic Effect of Ethanolic Extract of Mormodicalcharantial in Isoproterenol‐Induced Myocardial Infarction in Adult Wistar Rats
Authors: UBHENIN ABRAHAM EHINOMHEN , INNIH SILVANUS OLUSONYA
Published: June 2021

ISO was used to induce MI by intraperitoneal injection at the dose of 100mg/kg to Wistar rats Serum lipid profile and cardiac marker enzymes were obtained and then measured calorimetrically. The results show that isoproterenol‐induced myocardiac infarction were associated with significant (p<0.05) increase in the activities of cardiac marker enzymes in the serum with concomitant decrease in the activities of these enzymes in the myocardial tissue as compared to control group. There were also significant (p<0.05) increase in serum level of total cholesterol , trigyceride, low density lipoprotein and very low density lipoprotein in the group injected with isoproterenol ( group ii) as compared with control group . Pretreatment with leaf extract of Mormodicalcharantial significantly (p<0.05) prevented this alteration of the lipid profile and also of the activities of these cardiac marker enzymes both in the serum and myocardial tissue as compared to isopreterenol‐induced control group. Mormodicalcharantialpossesses cardioprotective effects.

Support or Funding Information

The authors receive no grant for this research but however are grateful to the Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Benin, for the use of their research laboratory also acknowledge the technical help of Prof. P.O. Udia , Dept. of Biochemisry, University of Benin, Benin City, Nigeria,

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Attenuation of FFA‐Induced Skeletal Muscle Insulin Resistance by Carnosic and Rosmarinic Acid
Authors: Hartogh Danja Den , Valvcheski Filip , Tsiani Evangelia
Published: June 2021

Impaired insulin action in muscle leads to insulin resistance and Type 2 diabetes mellitus, a disease on the rise. Elevated blood free fatty acids (FFAs), as seen in obesity, are associated with insulin resistance and studies have shown increased serine phosphorylation of the insulin receptor substrate (IRS‐1) and reduced insulin‐stimulated glucose uptake in muscle cells exposed to FFA palmitate. A number of serine/threonine kinases including JNK, IKKβ, mTOR and p70 6SK have been implicated in serine phosphorylation of IRS‐1 and insulin resistance. On the other hand, activation of the energy sensor AMP‐activated protein kinase (AMPK) increases glucose uptake and has become an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased skeletal muscle cell glucose uptake and activated AMPK. The polyphenols carnosic acid (CA) and rosmarinic acid (RA) are found in high concentration in RE and in the present study we investigated their effect on palmitate‐induced insulin resistant L6 muscle cells. Glucose uptake was measured using [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting.

Exposure of L6 myotubes to the FFA palmitate (P) (0.2 mM, 16 hours) resulted in significant reduction of insulin‐stimulated glucose uptake (I: 201±1.21% , P+I: 117±15.6% of control, P<0.001) indicating insulin resistance. Importantly, in the presence of 2 mM CA or 5 mM RA, the insulin‐stimulated glucose uptake was restored (CA+P+I: 185±7.8%, RA+P+I: 181±14.4% of control, P<0.001). Treatment with palmitate increased serine phosphorylation of IRS‐1, increased the phosphorylation/activation of JNK, mTOR and p70 6SK and significantly decreased the insulin‐stimulated phosphorylation of Akt. The effects of CA and RA on these signaling molecules are under investigation. Our data indicate that treatment with CA or RA attenuates the FFA‐induced muscle insulin resistance. These polyphenols may have potent effects against insulin resistance and deserve further study.

Support or Funding Information

The research was funded by the Natural Sciences and Engineering Research Council (NSERC) grant to ET.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Antidiabetic Effects of Hispidulin in Streptozotocin‐Induced Insulin Deficient Mice
Authors: Wang Yao , Alkhalidy Hana , Luo Jing , Liu Dongmin
Published: June 2021

In both Type 1 and type 2 diabetes, pancreatic beta‐cell failure plays an important role in the deterioration of glycemic control. In this study, we investigated whether hispidulin, a naturally occurring flavone, can be used to ameliorate diabetes in an animal model of insulin deficiency. Here, we report that oral administration of hispidulin (20 mg/kg) alleviates streptozotocin‐induced hyperglycemia in C57/BL6 male mice. Hispidulin effectively mitigates postprandial and fasting hyperglycemia and glucose tolerance, which was associated with higher circulating plasma insulin concentrations and improved glucose stimulated‐insulin secretion (GSIS). In addition, treatment with hispidulin reduced expression of pyruvate carboxylase and glucose 6‐phosphatase in the livers. In vitro, hispidulin suppressed glucose production in mouse primary hepatocytes, but potentiated GSIS in insulin‐secreting cells and mouse islets. These findings suggest that hispidulin could be a dual action, natural compound that exerts antidiabetic action via promoting beta‐cell function and suppressing hepatic glucose production.

Support or Funding Information

The work was supported by grants from NCCIH of NIH (1R01AT007077, 1R01AT007566)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Defining the role of CEBP/α in Functional β‐cell mass
Authors: Smith Courtn J , Leifer Aaron H , Ray Jason D. , Tessem Jeffery Sivert
Published: June 2021

Metabolic diseases, such as obesity and diabetes, continue to rise exponentially across the globe. More than 68.8% of adults are considered to be overweight or obese, with more than 14% of Americans suffering from diabetes. Prolonged states of hyperlipidemia and hyperglycemia are characteristic phenotypes of diabetes. Identifying genes involved in the regulation of blood glucose and adipogenesis are essential for establishing a more comprehensive treatment for diabetes. CCAAT‐enhancer binding protein‐α (CEBPα) has been shown to play a key role in hepatic regulation of blood glucose and adipogenesis. Inactivating CEBPα, results in dysregulation of adipose tissue browning and inhibition of glycemia regulation. These data demonstrate that CEBPα expression is necessary for energy metabolism. We have previously shown that the transcription factor Nkx6.1 is sufficient to upregulates genes necessary for inducing β‐cell proliferation, increasing cell survival and improving insulin secretion. Our previous microarray indicated a potential interaction between Nkx6.1 and CEBPα. We hypothesize that CEBPα is an intermediate which is necessary for Nkx6.1 to increase functional β‐cell mass. Here we demonstrate the effect of CEBPα mediated β‐cell proliferation, insulin secretion and cell survival. Furthermore, we demonstrate that knockdown of CEBPα downregulates c‐Fos, which is required for Nkx6.1 enhancement of functional β‐cell mass. These data demonstrate the role of CEBPα in increasing functional β‐cell mass.

Support or Funding Information

ADA 1‐17‐IBS‐101 to JST

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Susceptibility of Mice Lacking Renin‐b to Chronic Angiotensin II Infusion
Authors: Nakagawa Pablo , Nair Anand , Zhang Shao Yang , Lu Ko‐Ting , Zanaty Mario , Gomez Javier , Grobe Justin L , Sigmund Curt D
Published: June 2021

Previous experience to various hypertensive conditions enhances the response to subsequent hypertensive challenges. Importantly, the activation of the brain renin angiotensin system (RAS) is required for the sensitization to these hypertensive challenges. We previously reported that the brain‐specific renin isoform (Ren‐b) tonically inhibits the activation of the brain RAS and that ablation of Ren‐b (Ren‐b KO) results in brain RAS disinhibition leading to mild hypertension. Retrospective analysis of five cohorts of mice indicated that Ren‐b KO model exhibits a high degree of variability in blood pressure (BP) and heart rate, which may be attributed to the susceptibility to stress. Therefore, we hypothesized that Ren‐b KO mice are sensitized to a hypertensive challenge such as angiotensin II (Ang II). Wildtype (WT) or Ren‐b KO mice were infused with either vehicle or Ang II (400 ng/kg/min; sc) via osmotic minipumps for 4 weeks. All data was expressed as mean ± SE and unpaired t‐test was used for statistical analysis. P <0.05 was considered significant. Ang II significantly increased BP in both WT and Ren‐b KO to the same degree (WT+veh: 103±4 vs WT+Ang: 120±4 mmHg; p = 0.0143 (n=10–12) and KO+veh: 104±3 vs KO+Ang: 118±5 mmHg; p = 0.0291 (n=10–14)). Although the BP between Ren‐b KO and WT mice was elevated equally, Ang II induced an exaggerated suppression of plasma renin in Ren‐b KO compared to WT (WT+Ang: 14.8±2.1 vs KO±Ang: 7.2±2.6 ng/ml; p = 0. 0251; n=7–9). Ren‐b KO exhibited enhanced Ang II‐induced dipsogenic response (WT+Ang: 3.7±0.3 vs KO+Ang: 4.6±0.4 ml/day; p = 0. 0495; n=10–12) and exhibited elevated heart weight (WT+Ang: 4.8±0.2 vs KO±Ang: 5.2±0.1 mg/g of body weight; p = 0.026; n=10–12) concomitant with a 87% increase in cardiac collagen type I alpha‐2 mRNA expression (p = 0.0275; n=7–9). Altogether, this data indicates that in the presence of Ang II, Ren‐b KO exhibits susceptibility to cardiovascular disease including cardiac hypertrophy and fibrosis. After successive breeding and backcrossing Ren‐b KO mice at baseline developed a milder and more variable BP elevation compared to our initial cohorts. This suggests that epigenetic silencing or other physiological mechanisms, such as downregulation of peripheral RAS, might compensate for some, but not all, of the cardiovascular alterations driven by the genetic deletion of renin‐b. Studies of a conditional Ren‐b KO model will be used to elucidate the physiological and/or pathophysiological consequences of an acute downregulation of renin‐b in adulthood.

Support or Funding Information

NHLBI PPG P01 HL084207

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Acute and Chronic Anti‐Hypertensive Effects of Plantaginis Semen on Spontaneous Hypertension Rats
Authors: Chen Ping Chen , Lue Sheng‐I , Chen Sih‐Pei , Lu Tzong‐Shi , Weng Ching‐Feng , Fu Yaw‐Syan
Published: June 2021

Plantaginis semen (PS) is the seed of Plantago asiatica L., and it is widely used as a herbal medicine with antioxidant, diuretic and anti‐inflammatory effects. Hypertension is a cardiovascular disorder and it may lead to many cardiovascular diseases, such as stroke, cardiac hypertrophy, or heart failure. We focused on the acute and chronic anti‐hypertensive effects of Plantaginis semen extract (PSE) on spontaneous hypertensive rat (SHR) and our new inbred spontaneous phase‐1 hypertension (P1‐HT) modeling rats.

We used the rats with age of 8~10 weeks. Under anesthesia, continuous femoral arterial pressure of SHR and P1‐HT rats were recorded by intravascular catheter for 5 minutes prior the drug administration. In acute anti‐hypertensive effect test, the rats were orally administered with 0.36 g/kg/ml of PSE that homogenized and suspended in distilled water. The arterial pressure of each rat was consecutively recorded for more than 1hr after drug administration. In chronic anti‐hypertensive effect test, we orally administered the rats with 0.36 g/kg/ml per‐day of PSE for 4 weeks and measured blood pressure. We used Students–t test and one‐way ANOVA to test the effects of PSE treatment on blood pressure.

The pretreated mean arterial pressures of animals were 146±10mmHg in SHR rats, and 115±8mmHg in P1‐HT rats. The acute anti‐hypertensive effects of PSE could be observed within 15min after oral gavage and lasting more than 1hr. This acute effect of PSE could significant decrease the mean arterial pressure for 15mmHg on SHR rats, and 22mmHg on P1‐HT rats. The PSE treatment could slow down the heart rhythm significantly in both SHR and P1‐HT rats. The results of acute test of PSE indicate its anti‐hypertensive effects did not only act on diuretic pathway. In chronic tests, daily oral administrated with PSE did not change the pretreated reference value of blood pressure and its anti‐hypertensive effects.

In conclusion, our new inbred P1‐HT modeling rats are more sensitive than SHR rats on anti‐hypertensive test of PSE. It may be suitable for other anti‐hypertensive drugs development testing. Oral administrated with PSE may have a rapid anti‐hypertensive effect, and can last for a period of time. Daily administration with PSE can be considered as a treatment strategy for hypertension.

Support or Funding Information

NSYSUKMU107‐I001‐3

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
ATP release mediates the over‐activation of adaptive immune response associated with hypertension
Authors: Zhao Tuantuan , Li Yu , Bernstein Kenneth , Shen Xiao(Gregory)
Published: June 2021

The cause of most hypertensive disease is unclear, but inflammation appears critical in disease progression. However, how an elevated blood pressure (BP) initiates inflammation is unknown and the effects of high BP on immune responses are unfathomed. To study this, we immunized angiotensin II (Ang II) induced hypertensive mice and normotensive mice with ovalbumin (OVA) and adjuvants for 7 days. 2‐fold more OVA‐specific CD8+ T cells were present in the blood (2.7 ± 0.23% vs. 1.2 ± 0.47%, P<0.05) and the spleen (2.62 ± 0.31% vs. 1.31 ± 0.17%, P<0.05) of hypertensive mice as compared to normotensive animals. Two models of autoimmune disease were studied to determine if the exaggerated T cell responses in hypertension translate into increased tissue pathology. RIP‐mOVA is a transgenic mouse line that expresses OVA in pancreatic islet β cells. These mice were made hypertensive with either Ang II or L‐NAME. When OVA‐specific OT‐I T cells were infused into RIP‐mOVA mice, the hypertensive mice had significantly more OT‐I T cells infiltration into the pancreas (AngII 75%, L‐NAME 73%, control 13%) and developed severer diabetes (Blood glucose: AngII 331, L‐NAME 315, control 168 mg/dl). Concanavalin A induced hepatitis was also significantly worsened by hypertension as compared to the pathology observed in normotensive mice. A critical difference in hypertensive mice was the upregulation of CD86 on antigen presenting cells (APCs) (MFI:1928 ± 24.01 vs.1798 ± 20.96, P<0.05). Plasma ATP levels rose as early as 3 days after the induction of hypertension and reach a peak level of 3 μM after 2 weeks of hypertension, which exactly parallel the kinetics of CD86 expression on APCs. Hydrolyzing ATP or blocking the P2X7 receptor normalized CD86 expression and eliminated hypertension‐induced T cell over‐activation. Further, blind analysis shows untreated hypertensive patients have substantially elevated plasma ATP levels (2.21 ± 0.99 μM, n = 27) compared to treated hypertensive patients (0.94 ± 0.34 μM, n = 17) or normotensive controls (0.63 ± 0.38 μM, n =30). A linear trend was observed between ATP levels and systolic BP and diastolic BP among all subjects. These studies indicate ATP and CD86 as loci to potentially control hypertension associated inflammation.

Support or Funding Information

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.