bluuebunny/crossref_metadata_2025 huggingface dataset:
Does Coronary Artery Bypass Grafting Alone Correct Moderate Ischemic Mitral Regurgitation?
Authors: Aklog Lishan , Filsoufi Farzan , Flores Kathryn Q. , Chen Raymond H. , Cohn Lawrence H. , Nathan Nadia S. , Byrne John G. , Adams David H.
Published: July 2021

Background

The optimal management of moderate (3+ on a scale of 0 to 4+) ischemic mitral regurgitation (MR) remains controversial. Some advocate CABG alone, whereas others favor concomitant mitral annuloplasty. To clarify the optimal management of these patients, we evaluated the early impact of isolated CABG on moderate ischemic MR.

Methods and Results

Between January 1992 and August 1999, 136 patients (54% male, mean age 70.5 years, mean New York Heart Association class 2.7, mean ejection fraction 38.1%) with a preoperative diagnosis of moderate ischemic MR, without leaflet prolapse or pathology, underwent isolated CABG. Thirty-eight (28%) of 136 patients had intraoperative transesophageal echocardiography (TEE) before CABG, and 68 (50%) had postoperative transthoracic echocardiography (TTE) within 6 weeks of surgery. The subgroups of patients undergoing intraoperative TEE and postoperative TTE had preoperative characteristics similar to the overall group. The 30-day operative mortality was 2.9% (

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). Intraoperative TEE downgraded the severity of MR to mild or less (0 to 2+) in 89% (

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). On postoperative TTE, 40% (

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) continued to have at least moderate MR (3 to 4+), 51% (

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) improved somewhat to mild (2+) MR, and only 9% (

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) had resolution of their MR (0 to 1+). The mean preoperative, intraoperative, and postoperative MR grades were 3.0±0.0, 1.4±1.0, and 2.3±0.8, respectively (
P
<0.001).

Conclusions

CABG alone for moderate ischemic MR leaves many patients with significant residual MR and may not be the optimal therapy for most patients. Intraoperative TEE may significantly underestimate the severity of ischemic MR. A preoperative diagnosis of moderate MR may warrant concomitant mitral annuloplasty.
Abstract 564: Gender Dependent Protective Effects of Melusin Overexpression on Post Myocardial Infarction Remodelling and Survival in Mice
Authors: Unsoeld Bernhard , Teucher Nils , Neef Stefan , Vogt Johannes , Zyba Vitalij , Kumm Nina , Lau Eva , Knoell Ralph , Maier Lars S , Brancaccio Mara , Kaul Axel , Seidler Tim , Tarone Guido , Hasenfuss Gerd
Published: July 2021

This study is part of the EU funded EUGeneHeart consortium aiming at the identification of differential pathways leading from a compensated challenged heart to either beneficial or maladaptive hypertrophic signaling. A model for adaptive hypertrophy (swimming) was compared to two models of maladaptive hypertrophy (transverse aortic constriction, TAC and myocardial infarction, MI) in wildtype (WT) and melusin overexpressing (OE) mice with special attention towards gender differences. Melusin, a muscle specific β1 integrin interacting protein, was shown to favorably influence heart failure after TAC. To achieve the highest possible standardization and homogeneity the interventions were carried out at a single center (1423 mice) and the harvested samples were distributed among the consortium members for in-depth analyses of signaling pathways, energy metabolism as well as characterization of the extracellular matrix. Swimming (n=391) and TAC (n=426) lead to comparable and significant degrees of hypertrophy (heart weight/body weight ratio +13.3% and +9.7%, respectively). While TAC caused a concentric hypertrophy (LVEDD −8.2%) swimming significantly increased LVEDD by 9.3%. At this compensated stage there were no significant morphometric or echocardiographic differences between WT and melusin OE mice. However, following MI (n=606), melusin OE mice showed reduced dilatation (LVEDD +49.8% wt vs. +29.0% melusin OE, n=136) and wall thinning (septum thickness −40.5% WT vs. −21.4% melusin OE). Gender separated analysis revealed a significant reduction in LVEDD only in the melusin OE males. A significantly improved survival rate was found for the melusin OE males (59% vs. 37%) whereas there was no significant effect on survival in females (82% vs. 76%). Analysis of shortening and calcium transients indicated a still compensated myocyte function on the single cell level for all three models, as intended. This is the first study that shows a prognostic effect of melusin overexpression in the setting of MI. Reduced mortality is associated with more favorable remodeling. Most interestingly, the effect is gender dependent. The present findings may be of future clinical relevance. Examinations of signaling pathways are currently being performed.
Abstract 577: Origin and Therapeutic Efficacy of Human Cardiac Progenitor Cells
Authors: Mosna Federico , Bearzi Claudia , Rota Marcello , Hosoda Toru , Tillmanns Jochen , Yasuzawa-Amano Saori , Trofimova Irina , Siggins Robert W , LeCapitaine Nicole , D’Alessandro David A , Zias Elias , Quaini Federico , Urbanek Konrad , Rimoldi Ornella , Michler Robert E , Bolli Roberto , Kajstura Jan , Leri Annarosa , Frishman William H , Anversa Piero
Published: July 2021

To establish whether progenitor cells are present in the human heart (hCPCs) and have phenotypic properties distinct from human hematopoietic stem cells (hHSCs), samples of human myocardium were enzymatically dissociated and c-kit-positive-cells were sorted and plated immediately to obtain clones derived from single founder cells. By FACS analysis, freshly isolated hCPCs comprised 1.1±1.0% of the population and were negative for HSC markers, CD34 and CD133, and KDR. They were also negative for epitopes of monocytes, CD14 and CD16, mast cells, CD45, and lymphocytes, CD3 and CD20. The phenotype of hCPCs was completely different from that of human bone marrow cells which were positive for these surface antigens. Only small fractions of hCPCs expressed GATA4 and Nkx2.5. Of 1,530 seeded hCPCs, 11 clones were generated accounting for 0.7% cloning efficiency. Subsequently, these cells were injected in immunodeficient mice and rats at the time of coronary occlusion and 5 days after infarction. This was done to assess whether hCPCs differentiated into myocytes and coronary vessels immediately after ischemic injury and in the presence of a well-developed infarct. In both cases, hCPCs regenerated the infarcted myocardium. Connexin 43 and N-cadherin were detected between recipient rodent myocytes and newly formed human myocytes. The problem was then whether the structural integration of these two myocyte populations had a physiological counterpart. Studies were performed using an ex vivo preparation together with two-photon microscopy and laser line-scan imaging. EGFP-positive-hCPCs were injected in infarcted mice and the heart was studied 2-weeks later. The heart was perfused with an oxygenated Tyrode solution containing the calcium indicator Rhod-2 and stimulated at 1 Hz. Calcium transients was recorded in EGFP positive human myocytes and EGFP negative mouse myocytes. The synchronicity in calcium tracings between these two distinct cell pools was apparent, pointing to the functional integration of newly formed EGFP-positive human myocytes with the surrounding EGFP-negative mouse myocytes. Thus, the human heart contains hCPCs which are not of bone marrow origin and possess the ability to acquire the cardiomyocyte and coronary vascular cell lineages.
Abstract 590: Increased Endoplasmic Reticulum Stress in Atherosclerotic Plaques Associated With Acute Coronary Syndrome
Authors: Myoishi Masafumi , Minamino Testuo , Kitakaze Masafumi
Published: July 2021

Background
Endoplasmic reticulum (ER) responds to various stresses by up-regulation of ER chaperones, and prolonged ER stress eventually causes apoptosis. Although apoptosis is considered to be essential for the progression and rupture of atherosclerotic plaques, the influence of ER stress and apoptosis on rupture of unstable coronary plaques remains unclear.

Methods and Results
We obtained 152 coronary artery segments at autopsy and 40 atherectomy specimens from 71 and 40 patients, respectively . Smooth muscle cells (SMCs) and macrophages in the fibrous caps of thin cap atheroma and ruptured plaques, but not in the fibrous caps of thick cap atheroma and fibrous plaques, showed a marked increase in the expression of ER chaperone and numbers of apoptotic cells. ER chaperones also expressed higher in atherectomy specimens from patients with unstable angina pectoris than with stable angina. To explore the plausible molecular mechanism of activation of ER stress and the mechanistic link to apoptosis, we investigated plaque lipids such as oxysterols. Among oxysterols, expression of 7-ketocholesterol was increased in the fibrous caps of thin cap atheroma compared with thick cap atheroma. Treatment of either cultured coronary artery SMCs or THP-1 cells with 7-ketocholesterol induced upregulation of ER chaperones and apoptosis, while these changes were prevented by antioxidants. We also investigated possible signaling pathways for ER-initiated apoptosis and found that the CHOP (a transcription factor induced by ER stress)-dependent pathway was activated in unstable plaques. In addition, knockdown of CHOP expression by siRNA decreased ER stress-dependent death of cultured coronary artery SMCs and THP-1 cells.

Conclusions
Increased ER stress occurs in unstable plaques. Our findings suggest that ER stress-induced apoptosis of SMCs and macrophages may contribute to plaque vulnerability.
Abstract 168: Resveratrol Translocates Gsk-3β To Mitochondria And Protects The Heart At Reperfusion By Targeting The Mitochondrial Permeability Transition Pore
Authors: Xi Jinkun , Wang Huihua , Chanoit Guillaume , Cheng Guang , Mueller Robert A , Norfleet Edward A , Xu Zhelong
Published: July 2021

Although resveratrol has been demonstrated to be cardioprotective, the detailed cellular and molecular mechanisms that mediate the protection remain elusive. We aimed to determine if resveratrol protects the heart at reperfusion by modulating the mitochondrial permeability transition pore (mPTP) opening through glycogen synthase kinase 3β (GSK-3β). Resveratrol (10μM) given at reperfusion reduced infarct size (12.2 ± 2.5 % of risk zone vs. 37.9 ± 3.1 % of risk zone in control, n = 6) in isolated rat hearts subjected to 30 min regional ischemia followed by 2 h of reperfusion, an effect that was abrogated by the mPTP opener atractyloside (30.9 ± 8.1 % of risk zone), implying that resveratrol may protect the heart at reperfusion by modulating the mPTP opening. To define the signaling mechanism underlying the action of resveratrol, we determined GSK-3β activity by measuring its phosphorylation at Ser
9
. Resveratrol significantly enhanced GSK-3β phosphorylation upon reperfusion (225.2 ± 30.0 % of control at 5 min of reperfusion). Further experiments showed that resveratrol induces translocation of GSK-3β to mitochondria and translocated GSK-3β interacts with the mPTP component cyclophilin D but not VDAC (the voltage-dependent anion channel) or ANT (the adenine nucleotide translocator) in cardiac mitochondria. Taken together, these data suggest that resveratrol prevents myocardial reperfusion injury by targeting the mPTP opening via GSK-3β. Translocation of GSK-3β to mitochondria and its interaction with the mPTP component cyclophilin D may serve as an essential mechanism that mediates the protective effect of resveratrol on reperfusion injury.
Abstract 613: A Mass Spectrometric Analysis Of Embryonic Zebrafish Proteins
Authors: Lucitt Margaret B , Price Tom S , Pizarro Angel , Wu Weichen , Yocum Anastasia Yocum , Seiler Christoph , Pack Michael , Blair Ian A , FitzGerald Garret A , Grosser Tilo
Published: July 2021

Zebrafish is an attractive vertebrate model organism for studies into the molecular mechanisms of cardiovascular development, pathology and pharmacology. Studies into the genetics of protein expression are largely constrained by the availability of specific antibodies. Mass spectrometry based proteomics methods have the potential to overcome these hurdles. This requires firstly an accurate characterization of proteins accessible to targeted quantitative analysis. We applied mass spectrometric proteomic methodology and statistical analysis to create profiles of proteins expressed during zebrafish embryonic development. We detected 1307 proteins from 327,906 peptide sequence identifications at 72 hpf and 120 hpf with false identification rates of less than 1% using two dimensional chromatography tandem mass spectrometry. Close to two thirds of all detected proteins were derived from hypothetical or predicted gene models or were entirely unannotated. Comparison of protein expression in embryos by two dimensional gel electrophoresis differential in gel analysis (DIGE) revealed that proteins involved in energy production and transcription/ translation were relatively more abundant at 72 hpf consistent with the faster synthesis of cellular proteins during organismal growth. Pathway analysis revealed similar expression of proteins at both stages that relate to calcium, insulin receptor, ERK/MAP kinase, vascular epithelial growth factor signaling, and WNT/b-Catenin. Similarly both stages expressed proteins of the complement and coagulation cascades, GM-CSF, PTEN, and sonic hedgehog signaling and inflammatory signals. The data are accessible in a fully searchable database (http://bioinf.itmat.upenn.edu/zebrafish) that links protein identifications to existing resources including the Zebrafish Model Organism Database. This new resource should facilitate the selection of candidate proteins for targeted quantitation and may refine systematic genetic network analysis in vertebrate development and biology. This is the first large-scale proteome analysis of embryonic zebrafish tissue to reveal previously uncharacterized proteins and detect regulated proteins with relevance for cardiovascular function and development.
Abstract 620: Platelet CD40L: a Powerful Leukocyte and Endothelial Cell Activator in Atherosclerosis
Authors: Lievens Dirk , Zernecke Alma , Munnix Imke , Beckers Linda , Wijnands Erwin , Noelle Randy J , Heemskerk Johan W , Weber Christian , Daemen Mat J , Lutgens Esther
Published: July 2021

Here we investigated whether platelet CD40L plays a role in atherosclerosis. Platelets were obtained from donor ApoE−/− or CD40L−/− ApoE−/− mice, activated with thrombin, neutralized by hirudin and injected intravenously into n=30 ApoE−/− recipients (3E10
7
platelets) every 5 days. Infusion of activated CD40L−/− platelets in ApoE−/− mice from age 5–17wks caused a decrease in atherosclerosis (predominantly early lesions) in the aortic arch and its major branch points (ApoE−/− platelets 2.01E10
5
± 3.74E10
4
μm
2
vs CD40L−/−ApoE−/− platelets 1.01E10
5
± 1.57E10
4
μm
2
; p<0.05) to levels that were comparable to the sham (NaCl) group (1.17E10
5
± 1.61E10
4
μm
2
). To induce advanced atherosclerosis, a silastic non-constrictive collar was placed around both carotid arteries in diet fed mice, and activated platelets were injected every 5 days during 5 wks. Infusion of activated CD40L−/−ApoE−/− platelets caused a 35.4% decrease in plaque volume (ApoE−/− platelets 2.08E10
8
± 2.55E10
7
μm
3
vs CD40L−/−ApoE−/− platelets 1.36E10
8
± 3.45E10
7
μm
3
, Sham 1.17E10
8
± 2.71E10
7
μm
3
p<0.05), indicating an important role for activated platelet CD40L in both early and advanced atherosclerosis. Laminar flow assays showed that activated CD40L−/−ApoE−/− platelets lose their capacity of binding CD40L+/+ApoE−/− leukocytes, especially monocytes, with 30.1% compared to activated ApoE−/− platelets. Moreover, FACS analysis (CD45, CD11b,CD41) showed that activated ApoE−/−platelets bind 24.8% less to CD40−/−ApoE−/− leukocytes, and activated CD40L−/−ApoE−/−platelets bind 40.0% less to CD40−/−ApoE−/− leukocytes, thereby pointing out leukocyte CD40 as the receptor of platelet CD40L. Intravital microscopy in carotid arteries of mice, injected with activated CD40L−/− platelets, showed a 3-fold decrease in leukocyte adhesion. Even more, deficiency of CD40L on platelets causes a 58.4% decrease in thrombus formation compared to wild type. We demonstrate that CD40L on activated platelets plays an important role in atherosclerosis by promoting platelet aggregation, the formation of platelet-leukocyte aggregates, and leukocyte-endothelial interactions. This shows that platelet CD40L has a bridging function between the leukocyte and the endothelium.
Abstract 628: Pro-Apoptotic Bnip3 Functions as a Mitochondrial Sensor of Oxidative Stress in Myocardial Ischemia/Reperfusion
Authors: Kubli Dieter A , Quinsay Melissa N , Gustafsson Asa B
Published: July 2021

Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with mitochondrial dysfunction and cell death in the myocardium. We previously found that Bnip3 contributes to ischemia/reperfusion (I/R) injury, but it is not known how I/R causes activation of Bnip3. We have investigated potential mechanism(s) by which Bnip3 activity is regulated. Western blot analysis of heart lysates revealed that Bnip3 forms a ∼48 kDa complex after I/R that is sensitive to reduction by DTT. Complex formation was reduced when hearts were perfused with the reactive oxygen species (ROS) scavenger N-acetyl cysteine prior to I/R. The complex also increased in isolated myocytes treated with hydrogen peroxide, suggesting that the DTT-sensitive complex is formed by increased oxidative stress. To further investigate the function of this complex, we overexpressed Bnip3 in HL-1 myocytes and found that most of Bnip3 existed in the 48-kDa complex which correlated with increased cell death. In contrast, endogenous Bnip3 in the heart exists mainly as a monomer under normal conditions. Scanning of the Bnip3 protein sequence revealed a single conserved Cys residue at position 64, which may be susceptible to oxidation. Mutation of the Cys to an Ala or deletion of the N-term (aa 1–64) resulted in reduced cell death activity of Bnip3 compared to wild type when overexpressed in HL-1 myocytes. Separation of purified Bnip3 on SDS-PAGE showed the presence of the same DTT sensitive complex, suggesting that the complex is a Bnip3 homodimer. The transmembrane (TM) domain of Bnip3 has previously been identified to be important for homodimerization, and contains a His residue at position 173 that is essential for homodimerization. Mutation of the His to an Ala also resulted in reduced cell death activity of Bnip3 compared to wild type when overexpressed in HL-1 myocytes, suggesting that homodimerization is important for cell death activity of Bnip3. A consequence of I/R is the production of ROS and oxidation of proteins, which promotes formation of Cys disulfide bonds between proteins. Thus, these studies suggest that Bnip3 functions as a redox sensor in cells where increased oxidative stress induces homodimerization of Bnip3 via N-terminal Cys residue and the C-terminal TM domain.
Abstract 633: Suppression Of Mitochondrial GSK3β Activity By Its Ser9-phosphorylation Contributes To Cardiomyocyte Protection Afforded By Erythropoietin Against Oxidative Stress-induced Apoptosis
Authors: Ohori Katsuhiko , Miura Tetsuji , Tanno Masaya , Miki Takayuki , Satoh Takahiro , Horio Yoshiyuki , Shimamoto Kazuaki
Published: July 2021

Objective
Activation of the erythropoietin (EPO) receptor induces phosphorylation of numerous protein kinases, including glycogen synthase kinase 3β (GSK3β), and affords myocardial protection against ischemia/reperfusion injury. However, contribution of GSK3β to EPO-induced protection against apoptosis has not been directly demonstrated. Here, we examined whether alterations in activity and/or localization of GSK3β are critically involved in EPO-induced protection against oxidant stress-induced apoptosis.

Methods and Results
Apoptosis was induced in H9c2 cardiomyocyte by H
2
O
2
(50 μM) or angiotensin II (150 μM). Pretreatment with EPO (10 U/ml) suppressed H
2
O
2
-induced apoptosis (26.0± 2.7% vs. 38.3±2.9%, p<0.05) and also angiotensin II-induced apoptosis (21.5±0.9% vs. 31.9±1.4%, p<0.05) compared with that in untreated controls. Next, to examine the role of Ser9-phosphorylation of GSK3β, which suppresses GSK3β activity, H9c2 cells were transfected with EGFP-tagged wild-type (WT), constitutively active (S9A) or dominant negative (K85R) GSK3β. Apoptosis by H
2
O
2
was increased in S9A-transfected cells compared with that in WT-transfected cells (44.4±5.7% vs. 30.6±2.1%, p<0.05) and decreased in K85R-transfected cells (26.1±4.3%). EPO suppressed apoptosis in WT-transfected cells (21.9±2.5% vs. 30.6±2.1%, p<0.05) but not in S9A- or K85R-transfected cells. The effect of EPO was mimicked by suppression of GSK3β expression by its siRNA. Finally, to examine the effects of EPO on intracellular localization of GSK3β, H9c2 cells were transfected with WT and mitochondria were stained with MitoTracker. EPO did not change % of total GSK3β cololcalized with mitochondria but increased phospho-GSK3β colocalized with mitochondria by 55%.

Conclusion:
Together with reported inhibition of mitochondrial permeability transition by phospho-GSK3β, the present results suggest that suppression of GSK3β activity by its Ser9-phosphorylation in mitochondria plays a crucial role in cardiomyocyte protection by EPO against oxidant stress-induced apoptosis.
Abstract 639: Destabilization Of The Open State Of
I

Ks

Potassium Channels By A KCNQ1 V205M Missense Mutation Causes An Inherited Form Of LQTS In A Canadian Aboriginal Community
Authors: Rezazadeh Saman , Eldstrom Jodene , Rupps Rosemarie , Sanatani Shubhayan , Casey Brett , Tibbits Glen , Accili Eric , Arbour Laura , Fedida David
Published: July 2021

Hereditary long QT syndrome (LQTS) is a genetic disease characterized by a prolonged QT interval in the electrocardiogram and an increased likelihood of serious ventricular arrhythmias. We have determined that LQTS is disproportionately prevalent in a Northern British Columbia aboriginal community. Genetic screening of affected individuals revealed a novel missense mutation (V205M) in the S3 transmembrane helix of KCNQ1, the pore forming domain of the
I

K
s

channel complex. Co-expression of V205M KCNQ1 in mammalian cells with the accessory subunit KCNE1 recapitulated
I

Ks

, with similar surface expression to wild type (Wt) channels, but with a 33.7 mV depolarizing shift in the
V

1/2

of activation. This was accompanied by a slowing of channel activation (294 ± 85 ms, n=5; and 878 ± 124 ms, n=4; for Wt and V205M channels, respectively, at +70 mV), and an acceleration of deactivation (53 ± 3.6 ms, n=5; and 14 ± 3.5, n=4; for Wt and V205M channels, at −100 mV), determined at 35°C. The heterozygous state of the channel complex was simulated by co-expressing Wt and V205M channel subunits, and this resulted in phenotypic
I

Ks

currents with properties intermediate between Wt and V205M homomultimeric channels. Using a ventricular action potential voltage clamp protocol applied at 3 Hz, Wt channels, but not V205M channels, accumulated in the open state, which resulted in large outward
I

Ks

currents only for Wt channels. The outward ionic charge through heteromultimeric channels during the action potential clamp was reduced by more than 75% compared to Wt channels, suggesting a functional dominant negative effect of the V205M mutation at high heart rates. The changes in
I

Ks

kinetics produced by the V205M mutation are expected to decrease current and reduce the repolarization reserve during the cardiac ventricular action potential, with a likely increased susceptibility to the initiation of arrhythmias, especially during periods of high sympathetic drive.
Abstract 649: Does Chronic Increase of Glucose Uptake Cause Cardiac “glycotoxicity”?
Authors: Yan Jie , Luptak Ivan , Cui Lei , Jain Mohit , Liao Ronglih , Tian Rong
Published: July 2021

A shift of substrate preference toward glucose in the heart is considered a reversion to fetal metabolic profile but its role in the pathogenesis of cardiac diseases is incompletely understood. We performed a 2-year follow-up study in transgenic mice with sustained high glucose uptake and utilization in the heart by cardiac-specific overexpression of the insulin-independent glucose transporter GLUT1 (GLUT1-TG). Compared to wildtype (WT) littermates, the GLUT1-TG mice showed normal survival rate and unaltered contractile function of the heart monitored by serial echocardiography and by pressure-volume studies in isolated perfused hearts in the 2-year period. When the hearts were subjected to ischemia-reperfusion, an age-related impairment in functional recovery was observed in WT; cardiac function recovered to 35% vs. 52% of the preischemic level in old (22 months) vs. young (3 months) WT hearts respectively (p<0.05). Ischemic tolerance was markedly enhanced in GLUT1-TG hearts, and importantly, the greater functional recovery in GLUT1-TG hearts was sustained at older age (83% vs. 86% for old and young GLUT1-TG, respectively, p=ns).
31
P NMR spectroscopic measurement showed delayed ATP depletion, reduced acidosis during ischemia and improved recovery of high energy phosphate content in old GLUT1-TG hearts (p<0.05 vs. old WT). These differences were found to be independent of alterations in the activations of Akt and AMPK by ischemia. During reperfusion, glucose oxidation was 3-fold higher while fatty acid oxidation was 45% lower in old GLUT1-TG hearts compared to old WT (p<0.05) suggesting that the deleterious effects of excessive fatty acid oxidation during reperfusion was prevented in old GLUT1-TG hearts. Thus, these results suggest that a normal heart is capable of adapting to chronic increases in basal glucose entry into cardiomyocytes without developing “glucotoxicity”, and furthermore, life-long increases in glucose uptake result in a favorable metabolic phenotype that affords protections against aging-associated increase of susceptibility to ischemic injury.
Abstract 651: Myocardial NEFA Uptake is Enhanced to a Greater Extent Than NEFA Oxidation in Conscious Senescent Beagles with Myocardial Insulin Resistance
Authors: Bhashyam Siva , Brown Suzanne B , Friday Christine , Shorall Claire , Drabicki Brandy , Shannon Richard P
Published: July 2021

Background
: Prior studies have demonstrated that aging is associated with myocardial insulin resistance, mitochondrial ultra-structural changes and decreased UCP-3 protein. We sought to determine the relationship between these metabolic limitations and myocardial NEFA uptake and oxidation in senescent beagles.

Methods
: Eight young (age 3– 4 years) and 8 senescent (age 11–12 years) beagles underwent chronic instrumentation for measurement of hemodymics and myocardial substrate balance. All dogs were subjected to an 8 week exercise training program prior to instrumentation to control for activity level. In the fasting, conscious state, myocardial NEFA uptake and oxidation was measured using
3
H-oleate, and insulin resistance was assessed via hyperinsulinemic-euglycemic clamps. Samples of LV myocardium were harvested at euthanasia to measure components of NEFA transport.

Results
: There was no difference in body weight or adiposity between groups. Older beagles had marked (p<0.01) increases in plasma NEFA (Y:426± 65; O:902±78 μMol/min) and increased cardiac NE spillover (Y:21±4; O: 221±36 pmol/min). However, myocardial external efficiency was reduced (p<0.05) in older dogs (Y:15±3; O: 9±3 g·m
2
/ml O
2
/min). Hearts from older beagles demonstrated significant increases (p<0.01) in NEFA uptake (Y: 5.2±0.9; O: 9.3±0.8 μMol/min/100g), and only modest increases in oxidation (Y: 4.9±0.6; O: 7.0±1.1 μMol/min/ 100g). In sarcolemmal membranes, expression of Fatty Acid Transport Protein-1 (FATP-1) was upregulated (Y:72 11 DU; O: 235 21 DU, p<0.05), whereas that of FAT/CD36 was markedly reduced (Y: 1,312±123; O: 387±12 DU, p<0.001). There was no difference in PPARα (Y: 425±25; O: 475±116 DU) or PGC-1α (Y: 637±53; O: 633±14 DU) expression. The cumulative result of these changes was an accumulation of neutral lipids within older hearts and significant (p<0.02) insulin resistance (Y: 8.3 ±0.6; O: 4.3±0.8 μMol/min/100g).

Conclusion
: In older beagles, myocardial NEFA uptake exceeds oxidation, resulting in intramyocardial lipid accumulation. Increased FA uptake occurs despite decreased FAT/CD36 expression, and may be mediated by an upreguation of FATP-1 expression. These changes are seen in association with progressive myocardial insulin resistance.
Abstract 659: Simvastatin Inhibits Proliferation and Migration of Pulmonary Artery Smooth Muscle Cells from Patients with Idiopathic Pulmonary Arterial Hypertension
Authors: Nakamura Kazufumi , Ikeda Tetsuya , Fujio Hideki , Ogawa Aiko , Kobayashi Kaoru , Miura Aya , Miura Daiji , Nagase Satoshi , Fuke Soichiro , Oka Takefumi , Mohri Satoshi , Kusano Kengo F , Matsubara Hiromi , Miyazaki Masahiro , Date Hiroshi , Ohe Tohru
Published: July 2021

Background:

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease characterized by inappropriate increase of pulmonary artery smooth muscle cells (PASMCs) leading to occlusion of pulmonary arterioles and causing pulmonary hypertension. In this study, we assessed the inhibitory effects of simvastatin, an HMG-CoA reductase inhibitor, on proliferation and migration of PASMCs obtained from patients with IPAH.

Methods and Results:

PASMCs were obtained from 6 patients with IPAH who underwent lung transplantation, and PASMCs were obtained as control cells from 6 patients with bronchogenic carcinoma who underwent lung lobectomy. Platelet-derived growth factor (PDGF) (10 ng/mL) stimulation caused a significantly higher growth rate of PASMCs from patients with IPAH than that of control cells assessed by
3
H-thymidine incorporation (
P<
0.05). Simvastatin at 0.1 Â113>mol/L significantly inhibited PDGF-induced cell proliferation of PASMCs from IPAH patients but did not inhibit proliferation of control cells at the same concentration. Simvastatin at 1 Â113>mol/L also inhibited PDGF-induced migration of PASMCs from IPAH patients assessed by a transwell migration assay (
P<
0.05). Immunofluorescence staining revealed that simvastatin at 1 Â113>mol/L inhibited translocation of Rho A from the cytoplasm to membrane and disorganized actin fibers in PASMCs from IPAH patients.

Conclusions:

Simvastatin had an inhibitory effect on the inappropriate proliferation and migration of PASMCs from IPAH patients. Simvastatin may be useful for treatment of patients with IPAH.
Abstract 667: Angiotensin II-induced Osteopontin Expression in Vascular Smooth Muscle Cells involves G
q/11
, Ras, ERK, Src and Ets-1.
Authors: Abe Keiko , Ishida Mari , Sawano Mariko , Nakashima Hidekatsu , Soe Nwe Nwe , Hokama Lisa , Miho Narimasa , Sugino Hiroshi , Chayama Kazuaki , Yoshizumi Masao , Ishida Takafumi
Published: July 2021

Background
: Osteopontin (OPN), an extracellular matrix component produced by vascular smooth muscle cells (VSMC) and monocytes in response to biological stressors, is a regulator of cellular proliferation and migration. Recent studies revealed that OPN also plays a critical role in the progression of atherosclerotic plaques and that angiotensin II (AngII) is a potent upregulator of OPN expression. Therefore, the goal of the present study was to characterize the signaling mechanisms whereby AngII increases OPN expression.

Methods and Results
: YM254890, a specific inhibitor of G
q/11
, potently suppressed AngII-induced OPN expression and ERK1/2 activation. Among DN mutants of small G proteins (Ras, Rac, Rho), only DN-Ras completely suppressed AngII-induced OPN promoter activity. Dominant negative (DN)-MEK1 inhibited AngII-induced OPN promoter activity by 54%, while DN-c-Jun N-terminal kinase (JNK) or DN-p38MAP kinase had no effect. DN-Src and Csk suppressed AngII-induced OPN promoter activity by 49% and 71%, respectively. In addition, small interfering RNA against Ets-1 (a transcriptional factor downstream of ERK1/2) suppressed AngII-induced OPN expression by 54 ± 4.3%. In a separate experiment, rats were treated with the AngII type I receptor blocker, valsartan (1 mg/kg/day), or vehicle for 2 weeks after carotid artery balloon injury. The intima/media ratio and OPN expression were significantly lower in valsartan-treated rats than in vehicle-treated rats.

Conclusion
: These data suggest that AngII-induced OPN expression in VSMC is mediated by signaling cascades involving G
q/11
, the Ras-ERK axis, and c-Src, and by the transcription factor, Ets-1. Further, OPN may play a role in AngII-induced neointimal formation. These signaling molecules may represent therapeutic targets for the prevention of pathological vascular remodeling in patients with hypertension and atherosclerosis.
Abstract 668: Inhibition of DNA Methyltransferase Inhibits the Ang II-Induced Increase in Blood Pressure, Vascular Remodeling and Target Organ Damage
Authors: Bacanamwo Methode , Akinbami Mukaila , Wang Dahai , Gibbons Gary H
Published: July 2021

Growth signals stimulate DNA methyltransferase (DNMT) expression through extracellular signal-regulated kinases 1/2 (ERK1/2) pathway and that inhibition of ERK1/2 signaling results in repression of DNMT expression and activation of expression of some DNMT-repressed genes. Angiotensin II (Ang II) has been shown to stimulate ERK1/2 signaling in resistance arteries. We hypothesized that Ang II stimulates DNMT expression resulting in methylation and repression of antihypertensive genes such as 11β-hydroxysteroid dehydrogenase 2 (11β-HSD2) and that blocking DNMT activity may inhibit Ang II-induced hypertension, vascular remodeling, and target organ damage. C57BL/6J (C57) mice were treated with the specific DNMT inhibitor 5′-aza-2′-deoxycytidine (aza-CdR, 1 mg/kg/day) intraperitoneally for 3 consecutive days before subcutaneous Ang II infusion for 28d at a pressure dose of 0.7 mg/kg/day using Alzet osmotic minipumps. Systolic blood pressure was monitored during this period and tissues were harvested to determine vascular wall thickness, cardiac hypertrophy, and expression and methylation of 11β-HSD2 in arteries and the kidneys. Ang II infusion induced a more than 2-fold increase in DNMT1 expression (n=6, p≤ 0.05). Moreover, blocking DNMT1 activity significantly inhibited the Ang II-induced increase in systolic and diastolic blood pressure (from 102 ± 3 to138 ± 7 for saline-treated vs. 101 ± 4 to 105 ± 6 mm Hg for aza-CdR-treated mice, n=8, p≤ 0.01 for saline vs.aza-CdR in the presence of Ang II treatment). In addition, expression of 11β-HSD2 was inhibited by Ang II treatment. However, expression of 11β-HSD2 was found to be above the background observed in absence of Ang II when Ang II treatment was associated with DNMT activity blockade. Methylation-specific PCR indicated that 11β-HSD2 was methylated in the arteries and kidneys but its Ang II-dependency is still to be determined. Finally when DNMT activity was inhibited, the Ang II-induced increase cardiac hypertrophy (heart/body weight) and wall/lumen ratio in the carotid arteries were significantly inhibited (n= 8, p≤ 0.05). These results suggest that DNA methylation plays a critical role in the coordinate regulation of genes involved in the pathogenesis of hypertension and vascular remodeling.
Abstract 672: Long-Term Inhibition of Rho-kinase Ameliorates Diastolic Heart Failure in Hypertensive Rats
Authors: Fukui Shigefumi , Fukumoto Yoshihiro , Suzuki Jun , Saji Kenya , Nawata Jun , Tawara Shunsuke , Shinozaki Tsuyoshi , Kagaya Yutaka , Shimokawa Hiroaki
Published: July 2021

Background:

Diastolic heart failure (DHF) is a major cardiovascular disorder with poor prognosis; however, its molecular mechanism remains to be fully elucidated. We have previously demonstrated that activation of Rho-kinase pathway is substantially involved in the pathogenesis of oxidative stress and cardiac remodeling. In this study, we thus examined whether Rho-kinase pathway is also involved in the pathogenesis of DHF.

Methods:

We maintained male Dahl salt-sensitive rats for 10 weeks, starting from 7 weeks of age, with either 0.5% NaCl (control) or 8% NaCl (DHF) with or without oral treatment with fasudil, a selective Rho-kinase inhibitor (30 and 100 mg/kg/day) (n=13–31). We serially measured hemodynamic variables and cardiac function by echocardiography, followed by histological analysis.

Results:

At week 17, the untreated DHF group exhibited overt heart failure (e.g. lung edema) associated with diastolic dysfunction but with preserved systolic function, characterized by increased myocardial stiffness, cardiomyocyte hypertrophy, and enhanced cardiac fibrosis and superoxide production (all P<0.01 vs. control). By contrast, in the fasudil-treated group, those myocardial changes associated with DHF were significantly and dose-dependently ameliorated, independent of the changes in blood pressure, with a resultant inhibition of the transition to decompensated heart failure. Western blot analysis showed that cardiac Rho-kinase activity, as evaluated by the extent of phosphorylation of ERM, a downstream substrate of Rho-kinase, was significantly increased in the untreated DHF group and was dose-dependently inhibited in the fasudil-treated group. Importantly, there was a highly significant correlation between the extent of myocardial stiffness and that of cardiac Rho-kinase activity (R=0.74 for the posterior wall and R=0.63 for the septum, both P<0.001). Cardiac Rho-kinase activity also significantly correlated with cardiac superoxide production.

Conclusions:

These results indicate that activation of Rho-kinase pathway plays an important role in the pathogenesis of DHF through cardiomyocyte hypertrophy and interstitial fibrosis, suggesting the therapeutic importance of Rho-kinase for the treatment of DHF in humans.
Abstract 674: Coupling Factor 6 Activates c-Src By Intracellular Acidosis And Enhances Calcium Signaling In Resistance Arteriole Smooth Muscle Cells
Authors: Osanai Tomohiro , Tanaka Makoto , Tomita Hirofumi , Yamada Masahiro , Saitoh Sin , Kumagai Akiko , Murakami Reiichi , Ashitate Toshihiro , Echizen Takashi , Katoh Chisato , Yokoyama Hiroaki , Okumura Ken
Published: July 2021

In vascular endothelial cells, coupling factor 6 (CF6) binds to the β subunit of plasma membrane ATP synthase and attenuates prostacyclin generation by inducing intracellular acidosis. However, intracellular signaling in resistance arterioles that is directly related to vascular tonus and hypertension has not been determined yet. We investigated the direct effect of CF6 on Ca
2+
signaling, involvement of c-Src in it, and the role of CF6 in the pathogenesis of hypertension. CF6 at 10
−7
M induced a monophasic increase in intracellular free Ca
2+
concentration ([Ca
2+
]
i
) in A7r5 cells (rat vascular smooth muscle cells (VSMC) expressing voltage-gated Ca
2+
channel). When CF6 at 10
−7
M was added after pretreatment with nifedipine at 10
−5
M, the sustained increase in [Ca
2+
]
i
was abolished. In cultured resistance arteriole VSMC obtained from the mesenteric artery network of spontaneously hypertensive rats (SHR, n = 8) and Wistar Kyoto rats (WKY, n = 8), angiotensin II (Ang II) at 10
−7
M caused a spike increase in [Ca
2+
]
i
followed by a sustained increase. When Ang II at 10
−7
M was added after pre-administration of CF6 at 10
−7
M, the spike phase of [Ca
2+
]
i
was enhanced and it was greater in SHR than in WKY (412 ± 26 vs 162 ± 14 nM, p < 0.05). Pretreatment with PP1 at 50 μM, a c-Src inhibitor, blocked not only the CF6-induced Ca
2+
influx but the CF6-induced increase in the AngII-mediated spike phase of [Ca
2+
]
i
. CF6 receptor of VSMC was the β-subunit of ATP synthase, and the affinity was higher in SHR than in WKY (Kd: 3.6 ± 0.3 vs 9.6 ± 0.3 nM, p < 0.05). The increase in ATPase activity by CF6 was greater in SHR than in WKY (p < 0.05), and the decrease in intracellular pH was greater in SHR than in WKY (0.26 ± 0.03 vs 0.13 ± 0.02, p < 0.05). CF6 activated c-Src at 15 minutes by 2 folds greater in SHR than in WKY, and it was blocked by efrapeptine at 10
−5
M, an ATPase inhibitor. In mesenteric arterioles obtained from CF6-transgenic mice (2 fold over-expressed), AngII caused greater vasoconstriction by 2 folds than in those from wild mice (p < 0.05), but it was abolished by pretreatment with PP1. These suggest that CF6 activates c-Src by inducing intracellular acidosis and enhances Ca
2+
signaling in resistance arteriole VSMC. Since the c-Src activation was enhanced in SHR, CF6 might be involved in the genesis of hypertension.
Abstract 675: Contrasting Effects of Dietary Carbohydrate Compared to Mono-unsaturated Fat on Hepatic Production of ApoE and ApoC-III Containing VLDL, and Formation of LDL
Authors: Zheng Chunyu , Khoo Christina , Sacks Frank M
Published: July 2021

The effects of substituting dietary carbohydrate (CHO) with mono-unsaturated fat (MUFA) on plasma apoB metabolism were evaluated in 12 adults: 6 with normal and 6 with high plasma triglyceride levels. They consumed for 3 weeks each time a high CHO diet (48% complex CHO, 8% MUFA) and a high MUFA diet (31% complex CHO, 24% MUFA). ApoB100 kinetic studies were performed at the end of each dietary intervention using stable isotope labeling with a bolus and a primed continuous infusion. Multiple VLDL, IDL, and LDL fractions were prepared according to their apoE and apoC-III content. Compared to the CHO diet, the MUFA diet increased the percentage of VLDL and IDL secreted with both apoE and apoC-III (45% on MUFA vs. 14% on CHO, p < 0.01) and reduced the percentage of VLDL and IDL secreted without either apoE or apoC-III (MUFA 19% vs. CHO 40%, p = 0.02). Total liver secretion rates of apoB100 lipoproteins were similar between diets (MUFA 11.6 vs. CHO 11.3 mg·day
−1
·kg
−1
, p = NS). The dietary change did not affect the fractional catabolic rates and flux patterns of the lipoproteins. On both diets, VLDL and IDL that had apoE were rapidly cleared from the circulation, limiting LDL formation; whereas lipoproteins that did not have apoE or apoCIII mostly underwent lipolysis with little direct clearance, and were the main precursors of LDL. As a result, increased secretion of VLDL and IDL containing apoE and apoC-III caused by the MUFA diet was associated with higher direct clearance and lower LDL production rates (p = 0.02 vs. CHO), while the CHO diet increased LDL production due to increased secretion of VLDL without apoE or apoC-III. In conclusion, our results reveal a strong dietary effect on the secretion pattern of apoB100 lipoproteins. Substituting dietary complex carbohydrate with mono-unsaturated fat selectively promotes liver secretion of VLDL and IDL containing both apoE and apoC-III while suppressing the secretion of VLDL and IDL without apoE or apoC-III. This leads to significant downstream effects on LDL formation due to differential effects of apoE and apoC-III on apoB lipoprotein metabolism, resulting in enhanced particle clearance and reduced LDL formation with the MUFA diet compared to the CHO diet.
Abstract 677: The PPARdelta Agonist GW501516: Influence on Features of the Metabolic Syndrome
Authors: Sprecher Dennis L , Riserus Ulf , Hirschberg Sandra , Hassall David , Olson Eric J , Sarov-Blat Lea , O’Rahilly Stephen , Willson Timothy M , Murgatroyd Peter , Johnson Anthony , Frayn Keith , Karpe Fredrik
Published: July 2021

Hypertriglyceridemia and insulin resistance in the setting of obesity, i.e. the metabolic syndrome, are thought related to ectopic fat storage and impaired regulation of fat oxidation. Rodent models suggest that increased PPARdelta function may correct these abnormalities.

Design/Methods: Double-blind, randomized, 3-parallel group (n = 6 in each group), 2-week study of PPARdelta agonist (
GW501516
–10mg), PPARalpha agonist (
GW590735
–20ug) and placebo. Men with a waist > 95cm, BMI > 27, TG > 100mg/dl < 400mg/dl, and HDLc < 50 mg/dl were included. Blood lipids, fatty acids, serum insulin and liver fat content (MRI) were measured at baseline and termination of study, while oral
13
C-palmitate-labeled fat-feeding allowed post-meal examination of plasma NEFA metabolism and oxidation (
13
CO
2
exhalation). Skeletal muscle biopsies were taken to monitor transcript regulation of fat oxidation pathways.

Results: The PPARdelta group revealed reduction of LDLc, apoB and TG by 23%, 21%, and 31%, respectively and of plasma NEFA, without any deterioration of insulin and glucose homeostasis. Comparable TG reduction in PPARalpha (27%), with modest apoB reductions (−13%) were not accompanied by NEFA reductions. Hepatic fat was significantly reduced in the PPARdelta group only (p = 0.04). After the labeled fat meal, cumulative exhaled
13
CO
2
was increased (p = 0.03; enhanced fat catabolism) and accompanied by increased mRNA expression of skeletal muscle CPT1b only in the PPARδ group.

Summary: Short term administration of a selective PPARdelta agonist decreased TG, apoB, LDL-C and NEFA concentrations. Increased peripheral fatty acid uptake and oxidation may partly explain these benefits.
Abstract 175: Aldosterone Synthase Inhibition Prolongs Survival And Ameliorates Cardiac And Renal Dysfunction In An Angiotensin II-dependent Hypertensive Rat Model
Authors: Liu Jing , Chen Wei , Hu Chii-Whei , Jeng Arco Y , Rigel Dean F
Published: July 2021

Angiotensin II (Ang II) contributes to end-organ damage directly and also indirectly via stimulating aldosterone production. The present study evaluated the contribution of aldosterone to mortality/moribundity and cardiorenal injury in an Ang II-dependent model of hypertension (“double-transgenic rat”, dTGR). Aldosterone synthesis was blocked by pharmacologic inhibition of CYP11B2 (aldosterone synthase, AS) with FAD286 (FAD). FAD (1–100 mg/kg/day in drinking water from 3 to 8 weeks of age; n=10/group) dose-dependently prolonged median and 20% survival in dTGRs (Figure

). The optimal survival dose of FAD (30 mg/kg/day) was further evaluated in 7-week-old dTGRs (n=8), which were compared with age-matched untreated dTGRs (n=8) and untreated Sprague-Dawley (SD) rats (n=6). FAD improved cardiac systolic and diastolic function (echocardiography), attenuated cardiac hypertrophy and renal dysfunction, lowered plasma aldosterone concentration and urinary aldosterone excretion, but did not alter arterial pressure (Table

). Our results indicate that selective disruption of aldosterone biosynthesis prolongs survival and ameliorates organ damage.

This observation supports the concept that aldosterone is an independent cardiorenal risk factor and that AS inhibition may be a useful therapeutic approach.