bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P25: Hypothermia Treatment Inhibits Cardiomyocytes Calcium Overload and Mitochondria Transition Permeability after Hypoxia- Reoxygenation Injuries
Authors: Huang Chien-Hua , Hsu Chiung-Yuan , Tsai Min-Shan , Chen Huei-Wen , Cheng Hsiao-Ju , Pen Ren-How , Chen Wen-Jone
Published: July 2021

Introduction:
Hypothermia treatment can provide cardiac protection against ischemia reperfusion injuries, but underlying mechanisms remain unclear.

Hypothesis:
Hypothermia-related cardiomyocyte protection is through the mitochondrial dependent pathways.

Methods
: H9c2 rat cardiomyocytes were cultured in 37°C, 5% CO
2
incubators. After initiation of hypoxia-reoxygenation (H-R) treatment, the H9c2 cells were moved to hypothermia (31°C) or kept in normothermia (37°C) environments until cells harvested. Cell damage, intracellular and mitochondria calcium loads were studies. Mitochondria permeability transition and transmembrane potentials were studies by flowcytometric studies.

Results:
Hypothermia treatment ameliorates H9c2 cardiomyocytes survival after H-R injuries (68.1±11.8% vs. 85.0±12.7 %, P=0.025). Intracellular and mitochondria calcium overloading after H-R injuries was improved under 31°C environment (153.5±16.4 % vs. 957.1±311.7 %, P<0.01 for intracellular calcium; 101.8±28.5% vs. 159.4±32.5%, P=0.014 for mitochondria calcium). Mitochondria reductase activity were more preserved under hypothermia treatment after H-R injuries (55.7±10.9% vs. 8.5±1.2%, P<0.01). Hypothermia treatment decreased the continuous opening of mitochondria permeability transition pore after H-R damage by less reduction of mitochondria calcein fluorescence (15.6±13.7 % vs. 52.8±28.1 %, P=0.003). Release of cytochrome c into the cytoplasm from mitochondria after H-R injuries was more evident in normothermia condition by confocal microscopy study. Activation of caspase-9, which is down-streaming to cytochrome c, was down-regulated under hypothermia (62.1±21.9% vs. 87.5±7.3%, P=0.019). Loss of mitochondria integrity with decreasing of mitochondria transmembrane potential was less evident in 31°C than 37°C environments (55.9±23.3% vs. 102±20.2%, P=0.016).

Conclusion:
Hypothermia treatment at 31°C provides cardiomyocyte protection against hypoxia-reoxygenation injuries. The mechanisms are related to the decreasing intracellular and mitochondria calcium overloading, preserving the integrity of mitochondria by reduction of mitochondria permeability transition pore opening and cytochrome c release.
Abstract P167: Pentax-AWS is More Useful Than Macintosh Laryngoscope and Airtraq to Establish Tracheal Intubation while Uninterrupted Chest Compression: a Comparative Study with Manikin
Authors: Koyama Jun-ichi
Published: July 2021

Purpose. In resuscitation, chest compression is essential, and it should be performed appropriately and uninterruptedly to obtain the return of spontaneous circulation and breathing. However, attempt of tracheal intubation with the Macintosh laryngoscope (Mac) occasionally needs a temporary pause of chest compression and delays its resuming. Consequently, we studied the efficacy of the new video-laryngoscopes, PENTAX-AWS (AWS) and Airtraq (ATQ) as device used for tracheal intubation while nonstop chest compression.

Materials and Methods. Twenty seven participants including 11 non-anesthetic doctors, 1 paramedic, 11 nurses and 4 medical students enrolled in this study. At first, Participants and supplementary assistant who concentrated on chest compression simulated CPR with the use of Resusci Anne Simulator manikin (Laerdal) placed on medical emergency bed as 30:2 protocol proposed in 2005 AHA Guidelines. Strength and rhythm of the compression were monitored in real-time by PC skill-reporting system. After the chest compressions became constant, participants were requested to intubate with using Mac, AWS and ATQ in both situations without chest compression and while the compression.

Results. Tracheal intubation was successfully achieved within 3 attempts by 92.6% participants in Mac and 100% in AWS and ATQ without chest compression, and 66.7% in Mac, 100% in AWS and 81.5% in ATQ while chest compression. Rate of success in the 1st attempt was 55.6% in Mac, 96.3% in AWS and 51.9% in ATQ while compression. Time to intubate while compression with AWS was 14.6s, which was significantly shorter than 26.1s with ATQ in the successful attempts.

Conclusions. With using AWS, high success rate of tracheal intubation was achieved even in chest compression situation in comparison with the other two devices. Macintosh laryngoscope is the most inconvenient among these devices when tracheal intubation is attempted while chest compression. ATQ showed significant decrease of success rate in compression situation despite it achieved 100% success without compression. PENTAX-AWS is expected to be more useful than Macintosh laryngoscope and Airtraq to establish tracheal intubation while uninterrupted chest compression in clinical use.
Abstract 247: Therapeutic Neovascularization by AAV2/9 Based Gene Transfer of hVEGF-A and hPDGF in Chronic Ischemia (rabbit model)
Authors: Pfosser Achim , El-Aouni Chiraz , Globisch Franziska , Dinges Claudia , Fritz Andrea , Sultana Shahana , Hinkel Rabea , Lebherz Corinna , Banfi Andrea , Boekstegers Peter , Kupatt Christian
Published: July 2021

Therapeutic neovascularization by several growth factors is a concept well established in multiple animal models, albeit lacking clearcut clinical success to date. Therefore, we modified our therapeutic approach by combining the potent angiogenic agent hVEGF-A with hPDGF, a growth factor known for attracting pericytes and smooth muscle cells for arterial vessel maturation. In order to enhance efficacy, we utilized adeno-associated virus (AAV) 2/9, allowing for prolonged transgene expression. A lower hVEGF-AAV-concentration was used to avoid hemangiomaformation. 7 days after femoral artery excision, 1×10e11 hVEGF-A-AAV alone or in combination with 1×10e12 hPDGF-AAV particles were retroinfused into the anterior tibial vein of rabbits (n=5/group, p<0.05). LacZ-AAV2 was used as reporter-gene controls. At d7 and 35 angiography of both hindlimbs was performed for collateral quantification (% of d7 level) and frame count score (cinedensitometry, % of d7 level). Capillary density (Capillary muscle fiber ratio, CM/F) was assessed at d35 by PECAM-1 and alkaline-phosphatase-staining of the ischemic gastrocnemic and tibial anterior muscles, pericyte-quantification was assessed by NG2-staining. LacZ-expression was detected at d35 in control animals. hVEGF-A-AAV retroinfusion strongly induced angiogenesis (CM/F 1,32+0,07 vs. 0.96+0,08, controls), but failed to enhance collateral growth (125+7 vs. 95+6%, controls, p<0.05) or perfusion (frame count score: 136+12% vs. 107+9%, p=0.07). hVEGF-A/hPDGF co-application, however, enhanced perfusion (163+8%, p<0.05) via an increased collateral growth (146+9%, p<0.01) at a similar capillary density (1,44+0,10). We conclude, that efficacy of hVEGF-A-transfection is increased by hPDGF-co-transfection with a long-acting AAV. Utilizing this approach, the main target of hPDGF appears to be the collateral vasculature, complementary to the capillary growth induced by hVEGF-A. Thus, the combination of hVEGF-A and hPDGF combined with a long-acting AAV-vector is a promising tool in the quest of efficient therapeutic neovascularization.
Abstract 1444: Weighted Gene Co-expression Network Analysis of Adipose and Liver Reveals Gene Modules Related to Plasma HDL Levels and Containing Candidate Genes at Loci Identified in Genome Wide Association Studies
Authors: Langfelder Peter , Mehrabian Margarete , Schadt Eric E , Lusis Aldons J , Horvath Steve
Published: July 2021

The genetic and environmental factors contributing to HDL-cholesterol levels are highly complex. For example, a recent meta-analysis of three genome wide association studies (GWAS), consisting of over 9000 individuals, revealed several loci, but altogether these explained less than 10% of HDL variation. Since HDL has a heritability of about 50%, there clearly must be many as yet unidentified factors. To better address this complexity, we have utilized integrative genomic approaches to relate common DNA variation to gene networks and HDL metabolism. We report a Weighted Gene Co-expression Network Analysis (WGCNA) of genome-wide expression data from a CAST X C57BL6/J F2 intercross. WGCNA is a systems-based gene expression analysis and gene screening method. It utilizes co-expression patterns among genes to identify gene modules (groups of highly co-expressed genes) significantly associated with a clinical trait, in this case plasma HDL levels. Co-expression modules may represent cellular processes and interacting pathways that provide a bridge between individual genes and a systems-level view of the organism. A module-centric analysis effectively alleviates the multiple testing problems inherent in microarray data analysis and can be considered a biologically motivated data-reduction scheme. Using data from liver and adipose tissues, we have identified several modules strongly associated with plasma HDL levels (p-values ranging from below 1e-20 to 1e-5). Gene ontology and functional enrichment analysis indicate that these modules are indeed biologically meaningful. The modules contain variants of several genes under loci that were recently implicated by three GWA studies: liver modules include GCKR, ANGPTL4, ABCA3, APOA1, and APOA4, while the adipose modules include ABCA6, ANGPTL11 and 12, MMAB, MLXIPL, SORT1, PBX4, PLTP, and APOL6. Thus, our study also serves to help identify likely candidates from GWAS. In conclusion, applying WGCNA methods reveals modules that are biologically meaningful, statistically significant, and enriched for genes and pathways related to HDL metabolism and transport.
Abstract 1459: Tbx5 Regulates Progenitor Cell Migration During Cardiac Vasculogenesis
Authors: Nata Diman Y , Brooks Gabriel , Hatcher Cathy J , Burch John , Seidman John , Seidman Christine E , Basson Craig T
Published: July 2021

Mutations in TBX5 cause Holt-Oram syndrome (HOS) characterized by congenital heart malformation and upper limb deformity. Human TBX5 is expressed not only in a chamber specific myocardial distribution but also in coronary blood vessels. We have shown that mouse and chick Tbx5 are expressed in the proepicardial organ (PEO), a transitory structure from which epicardium and coronary vessels arise. We therefore investigated Tbx5 function during coronary vasculogenesis by manipulating Tbx5 gene dosage at critical points during chick and mouse cardioogenesis. Overexpression or inactivation of Tbx5 in chick PEO and chick epicardial cells leads to impaired migration and blocks incorporation of epicardial derived-cells (EPDCs) into coronary vessels. These data suggest Tbx5 acts during migration of PEO cells to the myocardium and migration of EPDCs into the subepicardium and myocardium. Epicardial restricted inactivation of Tbx5 using Wt1-cre and Tbx5 lox/lox mice reveals impaired migration (E10.5–11.5) of cytokeratin-positive PEO cells to the heart tube. However, at E14.5, mutant hearts display evidence of an epicardial layer suggesting delayed PE cell migration. However, unlike the flattened layer of epicardial cells in wildtype hearts, mutant hearts exhibit epicardial cells with irregular polygonal shapes and epicardial cyst-like structures lined by delaminated epicardium and filled with erythrocytes. At E17.5, we observe partial detachment of epicardium and nests of multiple contiguous subepicardial vessels without penetration into the myocardium. These observations suggest impaired migration of EPDCs into myocardium with consequent abnormally exuberant vasculogenesis restricted to the subepicardium. PECAM-1 expression analyses in E17.5 mutant embryos and neonatal mutant mice reveal marked reduction of myocardial coronary endothelial cells as well as defective coronary arteries and veins. These defects are associated with an upregulation of HIF1a in the myocardium, and, using an oxygen sensing dye, we observe relative tissue hypoxia in mutant myocardium. Our findings support a conserved requirement for Tbx5 in progenitor cell migration for the establishment of the coronary vasculature.
Abstract 1493: Albumin Excretion in Hypertensives with Sleep Apnea: Cross-Sectional Results in the Context of the Cross-Talk between Kidneys and Upper Airway
Authors: Thomopoulos Costas , Tsioufis Costas , Dimitriadis Kyriakos , Kasiakogias Alexandros , Stefanadi Elli , Makris Thomas , Tousoulis Dimitris , Stefanadis Christodoulos
Published: July 2021

Microalbuminuria reflects a state of widespread vascular dysfunction, whereas obstructive sleep apnea (OSA) further promotes atherosclerotic damage in hypertension. In a cross-sectional study, we investigated whether urinary albumin excretion, as expressed by urinary albumin to creatinine ratio (ACR) is increased in hypertensives with OSA compared to hypertensives without OSA. We studied 62 untreated hypertensives [aged 48±7 years, 49 men, 31 smokers, body mass index (BMI) 32±4Kgr/m
2
], with OSA documented by positive polysomnography [Apnea Hypopnea Index (AHI)>5] and 70 hypertensives without OSA (negative polysomnography), matched for age, sex, smoking status, BMI and 24h pulse pressure (PP). All subjects underwent polysomnography, 24h ambulatory blood pressure monitoring, echocardiography, routine biochemical assessment while the mean ACR of two non-consecutive morning spot urine samples was estimated. Hypertensives with OSA compared to those without OSA did not differ regarding 24h systolic BP (140±9 vs. 138±7 p=NS), while 24h diastolic BP and nighttime PP were higher in the former group (87±7 vs. 85±7 mmHg, p=0.03 and 50±10 vs. 45±10mmHg, p=0.008). Albuminuria as expressed by log10(ACR) was greater by 57% in patients with OSA compared to those without OSA (1.1±0.2 vs. 0.7±0.4mg/g, p<0.001). Metabolic profile and estimated glomerular filatration rate did not differ between the two groups (p=NS for all cases). In the entire study population log10(ACR) correlated with log10(AHI) (r=0.35, p<0.001), minimum oxygen saturation during sleep (r=−0.33, p<0.001), 24h PP (r=0.38, p<0.001) and nighttime PP (r=0.21, p=0.01). In a multivarible linear regression model including age, sex, BMI, smoking, GFR, glucose, LDL, 24h pulse pressure, and log10(AHI), independent predictors of ACR were AHI (β=0.36, p<0.001) and 24h PP (β=0.25, p<0.001). Albuminuria is increased within the normal range in hypertensives with OSA compared to those without OSA proportionally to OSA severity independently of confounders. The association of upper airway dysfunction with albuminuria and pultatile haemodynamic load may provide an explanatory mechanism for the OSA related risk in hypertension.
Abstract 1574: Low Myocardial Protein Kinase G Activity Raises Cardiomyocyte Resting Tension in Diastolic Heart Failure
Authors: van Heerebeek Loek , Hamdani Nazha , Handoko Martin L , van der Velden Jolanda , Stienen Ger J , Niessen Hans W , Laarman Gerrit Jan , Somsen Aernout , Paulus Walter J
Published: July 2021

Cardiomyocytes (CM) of diastolic heart failure (DHF) patients (pts) are hypertrophied and have high diastolic resting tension (RT), which elevates left ventricular (LV) diastolic stiffness. In rodent LV pressure overload models, development of pathological CM hypertrophy has been related to low myocardial protein kinase G (PKG) activity. Myocardial PKG activity and in-vitro effect of PKG on RT of isolated CM were therefore determined using LV endomyocardial biopsies procured from DHF pts. All DHF pts (n=36) had been admitted to hospital for worsening heart failure (NYHA 3– 4), were free of coronary artery disease, had a LV ejection fraction > 50%, a LV end-diastolic volume index < 97 ml/m
2
, a LV end-diastolic pressure > 16 mmHg and no histological evidence in the biopsies of myocardial infiltration or inflammation. Myocardial PKG activity was assessed by immunohistochemistry using antibodies against the specific PKG substrate vasodilator stimulated phosphoprotein (VASP) and against phosphorylated VASP ((P)-VASP). Myocardial PKG activity corresponded to the ratio of (P)-VASP/VASP. CM were isolated from the biopsies, treated with Triton X-100 to remove all membranes, attached to a force transducer and stretched to 2.2 μm to measure RT. In DHF pts, (P)-VASP/VASP ratio (0.67±0.15) was lower than in a control group (n=22) (0.98±0.19; p<0.0001). CM isolated from LV myocardium of DHF pts had higher RT (7.2±0.5 kN/m
2
) than CM of a control group (4.8±0.2 kN/m
2
; p<0.0001). In-vitro administration of PKG lowered RT of CM of DHF pts to levels similar to the control group (PKG: 4.0±0.7 kN/m
2
; p<0.01). Subsequent treatment with PKA did not further decrease RT (3.8±0.9 kN/m
2
). DHF patients had low myocardial PKG activity. This low myocardial PKG activity could have elevated RT of CM as in-vitro administration of PKG corrected the high RT. Absence of an additional in-vitro effect of PKA after PKG suggested that high RT results from hypophosphorylation of myofilamentary proteins, whose phosphorylation sites are targets for both PKA and PKG. In pts with DHF, phosphodiesterase 5A inhibition could raise myocardial PKG activity and thereby correct their high CM RT, which is an important determinant of their high diastolic LV stiffness.
Abstract 3490: Mechanistic Involvement of Myeloperoxidase in the Pathogenesis of Atrial Fibrillation
Authors: Rudolph Volker , Andrie Rene , Friedrichs Kai , Rudolph Tanja K , Klinke Anna , Lau Denise , Scoecs Katalin , Schrickel Jan , Lewalter Thorsten , Nickenig Georg , Meinertz Thomas , Willems Stephan , Baldus Stephan
Published: July 2021

Background: Observational clinical and ex-vivo studies have established a strong association between atrial fibrillation (AF) and inflammation. However, whether inflammation is cause or consequence of AF and which specific inflammatory mediators increase atrial susceptibility to fibrillate remain elusive. Herein, we provide evidence for mechanistic involvement of myeloperoxidase (MPO), a heme enzyme abundantly expressed by neutrophils, in the pathophysiology of AF.

Methods and Results: Patients with AF assessed by pacemaker interrogation not only exhibited higher circulating plasma levels of MPO (503.1 [IR:404.6 –880.7] vs. 437.8 [IR:348.9 – 488.0 pmol/l; p=0.03; n=42), they also revealed an increased MPO burden in explanted left atrial tissue as compared to patients devoid of AF. In AF-patients MPO co-localized with markedly increased formation of 3-nitro and 3-chlorotyrosin, protein oxidations known to be catalyzed by MPO. Myeloperoxidase knock-out mice, pretreated with angiotensin II infusion for 2 weeks yielding increased neutrophil activation, revealed strikingly attenuated vulnerability for AF during right atrial electrophysiological stimulation as compared to wild type mice (probability of AF-induction: 3.0 vs. 12.7%; p<0.01). Whereas the electrical homogeneity of the atrial myocytes was not altered between the groups, atria of MPO knock out mice were indicative of significantly reduced atrial fibrosis and markedly reduced formation of 3-chloro- and 3-nitrotyrosine.

Conclusion: In conclusion, the current findings not only underscore the significance of neutrophil activation as a critical pathophysiological prerequisite of AF, but reveal that MPO - by oxidatively modifying protein residues and increasing fibrosis of atrial myocytes - is causally linked to the initiation and perpetuation of AF.
Abstract 3684: Short-term Caloric Restriction Reverses Age-associated Vascular Endothelial Dysfunction in Older Mice
Authors: Rippe Catarina , Connell Melanie L , Lesniewski Lisa A , Donato Anthony J , Seals Douglas R
Published: July 2021

Backgound: Oxidative stress-mediated, nitric oxide (NO)-dependent vascular endothelial dysfunction develops with aging, increasing the risk of cardiovascular diseases. As such, it is important to identify interventions that can prevent/restore the loss of endothelial function with aging. Caloric restriction (CR) is associated with several physiological benefits and increased longevity in rodents. Some of these benefits are linked to increases in SIRT-1, an enzyme activating endothelial NO synthase (eNOS), and reductions in oxidative stress. We tested the hypothesis that short-term CR would restore vascular endothelial function by improving NO bioavailability and reducing oxidative stress in old B6D2F1-mice, and that this would be associated with increased expression of SIRT-1 and eNOS.

Methods: Old (30 months) male B6D2F1-mice were either fed ad libitum (AL, n=6) or were restricted to 70% (3.2g) of their normal food intake for 6 weeks after 2 weeks of progressive restriction (n=6). Carotid artery dilation was assessed ex vivo in response to the endothelium-dependent dilator acetylcholine (ACh) [before and after incubation with the eNOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) or the superoxide dismutase mimetic, TEMPOL] and the endothelium-independent dilator sodium nitroprusside (SNP).

Results: ACh-induced dilation was markedly impaired in the AL mice, but was completely restored in the CR mice (63±10% vs. 98±1%, P<0.01). L-NAME decreased ACh dilation by 97% in CR, but only 56% in AL mice, indicating increased NO bioavailability in the CR mice. TEMPOL restored ACh-mediated dilation in AL mice (to 97±1%), but had no effect in CR mice. Aortic protein expression of SIRT-1 and eNOS (western blotting) were 80 –90% higher in CR vs. AL mice (eNOS: 1.9±0.1 vs. 1.0±0.4 P<0.05; SIRT-1: 1.8±0.2 vs. 1.0±0.2, P=0.05). Dilation to SNP was similar in the CR and AL mice (97±1% vs. 96±2%, P=0.63).

Conclusion: Short-term CR selectively restores endothelium-dependent dilation in older B6D2F1-mice by increasing NO bioavailability and reducing oxidative stress. These effects are associated with increased vascular expression of SIRT-1 and eNOS. CR may be an effective intervention for reversing age-associated vascular endothelial dysfunction.
Abstract 3729: Scavenger Receptor Bi (sr-bi)-mediated Selective Uptake Is Required For The Remodelling Of Hdl By Endothelial Lipase
Authors: Nijstad Niels , Wiersma Harmen , Gautier Thomas , van der Giet Markus , Maugeais Cyrille , Tietge Uwe
Published: July 2021

Objectives

Endothelial lipase (EL) is a negative regulator of HDL cholesterol plasma levels, and scavenger receptor BI (SR-BI) is involved in remodelling of HDL. The present study investigates the requirement of SR-BI for the effects of EL-mediated phospholipid hydrolysis on HDL metabolism
in vivo
.

Methods and Results

In vitro
, selective uptake from EL-modified HDL was 129 % higher than selective uptake from control HDL in SR-BI overexpressing cells (p=0.01).
In vivo
overexpression of human EL by means of recombinant adenovirus decreased HDL plasma levels significantly (p<0.01). FPLC analysis and non-denaturing gel electrophoresis revealed that EL expression resulted in the generation of small HDL particles in wild-type mice, while in SR-BI
−/−
mice small HDL were preferentially removed. In kinetic experiments the fractional catabolic rate (FCR) of HDL cholesteryl ester (CE) increased by 110% (p<0.001) and the FCR of HDL apolipoproteins by 64% (p<0.001) in response to EL overexpression in wild-type mice. In SR-BI
−/−
mice a similar increase in the HDL apolipoprotein FCR occurred (p<0.001), however, no further increase in HDL CE catabolism. The apparent whole body selective uptake was increased 3-fold by EL in wild-type mice (p<0.001), while there was no selective uptake in SR-BI ko mice. EL overexpression increased hepatic selective uptake as well as holoparticle uptake (each p<0.01) in wild-type mice, while in SR-BI ko mice only holoparticle uptake increased (p<0.01).

Conclusions

Our results indicate that SR-BI-mediated selective uptake of HDL cholesteryl ester is essential for the remodelling of large alpha-migrating HDL particles by EL.
Abstract 3773: Myotrophin and the p53 Signaling Cascade in Cardiac Hypertrophy/Heart Failure
Authors: Das Biswajit , Young David , Vasanji Amit , Gupta Sudhiranjan , Popovic Zoran , Sen Subha
Published: July 2021

Myotrophin (Myo), a 12-kDa protein, stimulates myocyte growth and is a factor in initiating cardiac hypertrophy (CH). Cardiospecific overexpression of Myo in transgenic mice (Myo-Tg) induces hypertrophy that progresses to heart failure (HF). Oligonucleotide gene array revealed upregulation of a p53 homologue gene (EST-
AI843106
) in Myo-Tg mice during HF, indicating that p53 plays an important role during the transition of hypertrophy to HF. To dissect out the mechanisms of p53-mediated Myo-induced CH/HF, we developed a double-transgenic mouse line (p53
−/−
/myo
+/+
) by crossing Myo-Tg mice with p53-null mice. The double transgenic mice showed a significant attenuation of cardiac mass compared to Myo-Tg mice (heart weight:body weight ratio; 5.2 ± 0.21 vs. 7.9 ± 0.58,
p
< 0.001) associated with improved cardiac function and downregulation of ANF expression, suggesting that hypertrophy induced by Myo overexpression is indeed mediated through p53. To elucidate the relationship between p53 and Myo-induced hypertrophy, we performed a Reverse-Transcription Real-Time PCR pathway array on heart tissues from p53
−/−
/myo
+/+
vs. Myo-Tg mice. A bioinformatic approach, Ingenuity Pathway Analysis
TM
(IPA), was used to analyze the selected up-/downregulated genes. The IPA network showed that among the up-/downregulated genes,
Bcl2, Brca1, Cdkn1a
and
Myc
occupy the nodal position, whereas
E2f1
,
Pmaip1
,
Gadd45a
and
Pttg1
function as peripheral candidates. The expression profiles of some genes of the p53 pathway were validated by immunoblot analysis. Functional assignment of these selected candidate genes showed that
Bcl2, E2f1
and
FasL
are related to CH/HF, but the function of
Gadd45a, Pmaip1,
and
Vcan
is still unknown. Apart from these p53 cascade members, we also found that other molecules (e.g., Jnk, Ras, NF-kB, Cyclin L, and Mek) may be involved in an intricate interplay to stimulate p53-mediated Myo-induced CH. Suppression of NF-kB activity (by electrophoresis mobility shift assay) in p53
−/−
/myo
+/+
mice compared to Myo-Tg mice indicated involvement of NF-kB, as predicted by IPA, in Myo/p53 cross-talk. Our data suggest that the p53 signaling cascade actively participates in progression of hypertrophy to HF, triggered by overexpression of myotrophin.
Abstract 3782: β2-Adrenergic Receptor Signaling Positively Modulates Pro-Survival Kinases and Ameliorates Mitochondrial Dysfunction during Doxorubicin Cardiotoxicity
Authors: Fajardo Giovanni , Zhao Mingming , Berry Gerald , Mochly-Rosen Daria , Bernstein Daniel
Published: July 2021

β2-adrenergic receptors (β2-ARs) modulate cardioprotection through crosstalk with multiple pathways. We have previously shown that β2-ARs are cardioprotective during acute exposure to Doxorubicin (DOX). DOX cardiotoxicity is mediated through a Ca
2+
-dependent opening of the mitochondrial permeability transition pore (MPT) and mitochondrial dysfunction, however the upstream signals linking cell surface receptors and the MPT are not clear. The purpose of this study was to assess crosstalk between β2-AR signaling and mitochondrial function in DOX toxicity. DOX 10 mg/kg was administered to β2−/− and WT mice. Whereas there was no mortality in WT, 85% of β2−/− mice died within 30 min (n=20). Pro- and anti-survival kinases were assessed by immunobloting. At baseline, β2−/− showed normal levels of ϵPKC, but a 16% increase in δPKC compared to WT (p<0.05). After DOX, β2−/− showed a 64% decrease in ϵPKC (p<0.01) and 22% increase in δPKC (p<0.01). The ϵPKC activator ΨϵRACK decreased mortality by 40% in β2−/− mice receiving DOX; there was no improvement in survival with the δPKC inhibitor δV1–1. After DOX, AKT activity was decreased by 76% (p<0.01) in β2−/− but not in WT. The α1-AR blocker prazosin, inhibiting signaling through Gαq, restored AKT activity and reduced DOX mortality by 47%. We next assessed the role of mitochondrial dysfunction in β2−/− mediated DOX toxicity. DOX treated β2−/− mice, but not WT, show marked vacuolization of mitochondrial cristae. Complex I activity decreased 31% in β2−/− mice with DOX; but not in WT. Baseline rate of Ca2+ release and peak [Ca2+]i ratio were increased 85% and 17% respectively in β2−/− myocytes compared to WT. Verapamil decreased mortality by 27% in DOX treated β2−/− mice. Cyclosporine, a blocker of both MPT and calcineurin, reduced DOX mortality to 50%. In contrast, FK506, a blocker of calcineurin but not the MPT, did not reduce DOX mortality. Cyclosporine prevented the decrease in AKT activity in β2−/− whereas FK506 did not. These findings suggest that β2-ARs modulate pro-survival kinases and attenuate mitochondrial dysfunction during DOX cardiotoxicity; absence of β2-ARs enhances DOX toxicity via negative regulation of survival kinases and enhancement of intracellular Ca2+, sensitizing mitochondria to opening of the MPT.
Abstract 3877: The Magnitude and Temporal Dependence of Apoptosis Early After Myocardial Ischemia with or without Reperfusion
Authors: French Christopher , Zaman A.K.M. Tarikuz , Spees Jeffrey L , Taatjes Douglas J , Sobel Burton E
Published: July 2021

It has been hoped and conjectured that in addition to improving the balance between myocardial oxygen supply and demand amelioration of apoptosis could preserve jeopardized ischemic myocardium destined to undergo necrosis (cell death). Assessment of apoptosis in experimental animals has been based generally on TUNEL assays. Unfortunately, TUNEL positivity occurs with oncotic, mitotic, and autophagic, as well as apoptotic cell death. Accordingly, we characterized the extent of apoptosis after transient or persistent ischemia with highly specific methods. Coronary artery occlusion with and without subsequent revascularization was induced in three groups of 10 week old C57BL/6 mice: those subjected to 1 hr or 4 hr transient ligation followed by 24 hr of reperfusion (1HTLR24H, 4HTLR24H); or 24 hr persistent ligation (24HPL). Apoptosis was quantified throughout the LV by TUNEL, single stranded DNA (ssDNA), and caspase 3 immunohistochemistry, electron microscopy (EM), and caspase 3 and 8 activities assessed biochemically. TUNEL staining markedly exceeded and did not correlate with ssDNA, caspase 3 staining, or apoptosis defined by EM in any group. It was lowest in the 1HTLR24H group (averaging 38.6 of cells ± 3.1% [SEM], n = 9) greater in the 4HTLR24H group (48.5 ± 3.1%, n = 9), and significantly greater than both in the 24HPL group (67.2 ± 4.3%, n = 9, p < 0.01). ssDNA staining was minimal and similar in all three groups and significantly less than TUNEL staining (p < 0.01) (1HTLR24H, 1.0 ± 0.2%, n = 9; 4HTLR24H, 0.8 ± 0.1%, n = 9; 24HPL, 2.9 ± 1.6%, n = 9). Caspase 3 activity/mg protein (1HTLR24H, 15.3 ± 12, n = 3; 4HTLR24H, 58.5 ± 4, n = 3; 24HPL, 56.5 ± 10, n = 3) was similar to that in normal hearts (53.2 ± 11, n = 4), as was caspase 8 activity. No cleaved caspase 3 was seen immunohistochemically, and only rare, definitively identified apoptotic cells were seen by EM in hearts from any group. Apoptosis is de minimus early after transitory or persistent ischemia contrary to its overestimated, large extent as judged from TUNEL. Thus, antiapoptotic interventions per se are not likely to preserve substantial amounts of jeopardized ischemic myocardium early after ischemic insults.
Abstract 3968: Bone Marrow Derived Growth Differentiation Factor-15 (GDF-15) Protects from Macrophage Accumulation and Effects Atherosclerotic Lesion Stabilisation in Low-Density Lipoprotein Receptor-Null Mice
Authors: Preusch Michael R , Strelau Jens , Baeuerle Matthias , Blessing Erwin , Bischof Marc , Kinscherf Ralf , Unsicker Klaus , Katus Hugo A , Bea Florian
Published: July 2021

Atherosclerosis is considered to be a chronic inflammatory disease. Macrophages are the prime sources of a variety of inflammatory cytokines and growth factors, which contribute to the initiation and progression of atherosclerotic lesions. The cytokine growth differentiation factor-15 (GDF-15) is a newly discovered member of the transforming growth factor-beta cytokine. GDF-15 participates in vascular inflammation and is mostly expressed by macrophages within the lesions. In this study the impact of GDF-15 deficiency in bone marrow-derived cells on atherogenesis in a mouse model was examined. Bone marrow from GDF15 −/−or GDF-15 +/+ mice was transplanted into lethally irradiated low-density lipoprotein receptor (LDLR−/−) mice (n=38). Twentyfour weeks after administration of a high-fat/high-cholesterol Western type diet atherosclerotic lesion size within the aortic root as well as macrophage content was quantified and compared. In addition features of lesion destabilisation like size of the necrotic core, thinning of the fibrous cap, intra-plaque hemorrhage and calcification were evaluated. In an in-vitro experiment peritoneal macrophages from transplanted mice were harvested and stimulated with tumor necrosis factor alpha (TNFα). Transplantation of GDF-15 −/− bone marrow cells resulted in an enhanced macrophage accumulation within the atherosclerotic lesions (ratio mac/lesion 0.51 versus 0.31; p<0.05) and a significant thinning of the fibrous cap (30.5 versus 48.5 μm; p<0.05). Cell culture experiments demonstrated that macrophages from GDF-15 −/− mice had a much higher induction of ICAM-1 and MCP-1 after stimulation with TNFα in comparison to wildtype peritoneal macrophages. However no difference in lesion size could be reported. Furthermore, there was no difference in plasma lipid levels and body weight. Our data indicates that bone marrow derived GDF-15 protects from macrophage accumulation within atherosclerotic lesions and promotes lesion stabilisation possibly due to inhibition of adhesion molecules and MCP-1.
Abstract 5309: beta-Arrestin 1 (bArr1) and Ankyrin 2 (ANK2) Regulate Cav1.2 Trafficking through Complex G Protein Mediated Signaling
Authors: Diverse-Pierluissi Maria A , Lipsky Rachele , Puckerin Akil A , Stocks Joanne , Patel Vivek , Akar Fadi G
Published: July 2021

Mechanisms governing the trafficking of cardiac voltage dependent calcium channels (Ca
v
1.2) remain largely unknown. We have previously shown that neuronal calcium channels are internalized into clathrin coated vesicles upon G protein coupled receptor (GPCR) activation. Here, we hypothesize that GPCR mediated signaling regulate Ca
v
1.2 trafficking and surface expression through interaction with cytoskeletal and signaling molecules.

Methods
: Sequential immunoprecipitation in adult rat cardiomyocytes was used to investigate the interaction between Ca
v
1.2, cytoskeletal and signaling molecules. Live cell imaging and immunofluorescence microscopy were used to measure changes in the cellular localization of Ca
v
1.2 and these proteins upon activation of GPCR signaling.

Results
: Cav1.2 interacts with ANK2, βArr1 and spectrin. Sustained β adrenergic receptor (βAR) activation increases Cav1.2/spectrin and decreases Ca
v
1.2/ANK2 and Ca
v
1.2/βArr1 interactions. Sustained βAR activation induces rapid (<5 min) Cav1.2 internalization and dissociation from ANK2 as Ca
v
1.2/ANK2 co-localization is markedly reduced (−58% p<0.01) in isoproterenol treated myocytes. A cell permeant peptide (1.4 μg/ml) that disrupts Ca
v
1.2/ANK2 interaction decreases (−40±12%, p<0.01) Cav1.2 cell surface expression. Pretreatment with pertussis toxin prevents Ca
v
1.2 internalization suggesting that G
i/o
mediates this response. Similarly, Src kinase inhibition reduces (by 90%) Ca
v
1.2 internalization. Differential labeling of pre-existing Ca
v
1.2 at the cell membrane and newly inserted Ca
v
1.2 using fluorophore conjugated dihydropyridines reveal that activation of G
i/o
proteins underlies Ca
v
1.2 internalization and insertion. In contrast, activation of the muscarinic M2 receptor (10μM carbachol) causes only insertion of new channels (n=14) and fails to alter the association of Ca
v
1.2 with ANK2 and spectrin. Finally PI3 kinase inhibition (10nM wortmannin) prevents Ca
v
1.2 membrane insertion.

Conclusions
: Our findings reveal novel signaling mechanisms that regulate Ca
v
1.2 trafficking, internalization and membrane insertion in a receptor subtype dependent manner. These mechanisms may be central to pathologies associated with a hyperadrenergic state.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5482: Reduction of Established Atherosclerosis in Hypercholesterolemic Mouse Models by ATI-5261: a Novel α-helix Peptide that Stimulates ABCA1 Cholesterol Efflux with High Efficiency
Authors: Bielicki John K , Zhang Haiyan , Azhar Rakia , Johansson Jan , Azhar Salman
Published: July 2021

Apolipoprotein(apo)s of HDL stimulate cholesterol efflux via the ATP-binding cassette transporter A1 (ABCA1), generating HDL and reversing the macrophage foam-cell phenotype. Despite these anti-atherogenic roles, the impact of specifically promoting ABCA1 cholesterol efflux on atherosclerosis development is largely unknown. Progress in this area has been hindered by a lack of synthetic agonists and/or apo mimetics optimized to mediate ABCA1 cholesterol efflux. The latter can be attributed, in part, to the fact that long segments of α-helical structure constituting the C-terminal (CT) domains of apoA-I and E are required to mediate ABCA1 cholesterol efflux efficiently. To begin to address these issues, we compressed the activity determinants of these CT helices into a single α-helix, creating a peptide (ATI-5261) that stimulates ABCA1 efflux with apo molar efficiency. Treatment of LDLr−/− mice made atherosclerotic by a high-fat western-diet (13 weeks) with ATI-5261 (30 mg/kg)
ip
on alternate days for 6 weeks reduced atherosclerosis by ~30%, as judged by lesion-area (Sudan IV) covering the aorta (7.9±2 vs.11.3±2.5% control, p=0.011), and the lipid-content (oil-red O) of aortic-sinus plaque (25±5.8 vs. 33±4.9% control, p=0.034). In similarly designed studies, ATI-5261 reduced atherosclerosis in apoE−/− mice by ~45% (lesion-area = 14±8 vs. 25±9% control, p=0.011; plaque lipid-content = 22±6 vs. 41±7% control, p=0.001). The minimum effective dose of ATI-5261 was 3 mg/kg in apoE−/− mice, consistent with the potent cholesterol efflux activity of the peptide. In both mouse models, ATI-5261 reduced atherosclerosis to the same extent as ATI-5261:POPC complexes (~7 nm). Single ip injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces for up to 48 h. To our knowledge, ATI-5261 is the first synthetic α-helix able to reduce atherosclerosis in mice having substantial plaque-lipid accumulation. This did not require peptide formulation with phospholipids and was achieved in the presence of vastly different lipoprotein profiles and plasma cholesterol concentrations. The data suggest that accelerating ABCA1 lipid-efflux may represent a viable approach to reduce atherosclerosis and stabilize vulnerable plaque.
Abstract 5573: Notch Signaling Enhances BMP2-induced Msx2 Gene Expression and Vascular Calcification
Authors: Shimizu Takehisa , Tanaka Toru , Iso Tatsuya , Kurabayashi Masahiko
Published: July 2021

Background:
Bone morphogenetic protein2 (BMP2) and transcription factor Msx2, known as major regulators of osteoblastic differentiation, have been implicated in the pathogenesis of vascular calcification. Recent evidences indicate that Notch signaling regulates valve calcification and atherosclerosis. In this study, we examined whether Notch signaling effects on BMP2-induced Msx2 gene expression and vascular calcification.

Methods and Results:
Treatment with either BMP2 or adenovirus expressing Notch intracellular domain (NICD), slightly induced the expression of the Msx2 gene in pluripotent mesenchymal stem cells and human aortic smooth muscle cells. Surprisingly, either NICD overexpression or stimulation by Notch ligands markedly augmented the Msx2 gene expression in the presence of BMP2, as well as other osteogenic genes expression. A knockdown of Msx2 expression using siRNA drastically attenuated synergistic ALP activity induced by BMP2 and NICD. Luciferase assay showed transcription activity of Msx2 promoter, which contains Smad binging elements (SBE) essential for BMP2 responsiveness, was synergistically enhanced by Notch ligands and BMP2 treatment, whereas this synergism was almost completely inhibited in the presence of Notch signaling blocker. Interestingly, DNA affinity precipitation assay revealed that NICD-RBPJK complex bound to Msx2 promoter very adjacently to SBE, which indicates that RBPJK-bound NICD might interact with Smads and this interaction might lead to enhanced BMP2 signaling and Msx2 expression. Correspondingly, OT11 cells, in which RBPJk expression was deficient, did not show increased Msx2 activation in response to BMP2 and Notch signaling.

Conclusion:
These results suggest that BMP2 and Notch signaling cooperatively promote osteogenic differentiation which may mediate vascular calcification in the development of atherosclerosis.
Abstract 2007: Effects of Myocardial Viability Assessed by Strain Stress Echocardiography on Diastolic Function in Acute Myocardial Infarction
Authors: Chung Wook-Jin , Shin Mi-Seung , Bong Jeong Min , Kang Woong Chol , Han Seung Hwan , Moon Chan Il , Koh Kwang Kon , Ahn Tae Hoon , Choi In Suk , Shin Eak Kyun
Published: July 2021

Purpose:
Myocardial viability may influence the diastolic function of left ventricle (LV) reflecting chamber remodeling in the early phase after acute myocardial infarction (AMI). However, the precise relationship in human has not been determined yet. So we aimed to evaluate whether myocardial viability assessed by strain rate parameters on low-dose dobutamine echocardiography (LD-DSE) may influence on LV diastolic function and remodeling process in AMI.

Methods:
In this prospective single center study, 134 consecutive patients with AMI and akinetic wall motion in at least two segments were enrolled and underwent LD-DSE on 5~9 days after the event. Myocardial viability was quantitatively evaluated by global peak systolic strain rate (psSR) and global time to peak of psSR. Within 6 hours after LD-DSE, left and right heart catheterization with the recording of LV end diastolic pressure and pulmonary capillary wedge pressure were performed. And then 6 months later, echocardiography and thallium scan with delayed image were followed.

Results:
Patients (67.9% men, mean age 60.1±1.1years, 54.5% anterior wall, 53.7% ST-segment elevation) were tolerated LD-DSE without significant complications. 71.6% of patients underwent revascularization therapy. After 6 months later, LV ejection fraction was significantly increased from 48.7±1.1% to 52.0±1.1% (p=0.00) and LV end diastolic volume was significantly decreased from 94.6±4.0cc to 89.1±3.8cc (p=0.00). As we expected, global psSR at baseline of LD-DSE showed significant linear correlations with LV end diastolic pressure, E/E’ ratio and left atrial volume index (r=−0.43 and p=0.01, r=0.52 and p=0.00 and r=0.52 and p=0.00, respectively). And also global psSR at 10mcg of LD-DSE correlated with E/E’ ratio and left atrial volume index(r=0.52 and p=0.00 and r=0.36 and p=0.02, respectively). Interestingly, global psSR at 10mcg of LD-DSE correlated significantly with the change of LV end diastolic volume during 6 months follow-up (r=−0.42 and p=0.04).

Conclusions:
Strain rate data on LD-DSE correlates with LV diastolic function in the early phase and chamber remodeling in 6 months follow-up after AMI. So strain stress echocardiography may provide useful prognostic information for LV remodeling in AMI.
Abstract 2089: Randomized Controlled Trial of the Effects of Lifestyle Modification on Metabolic Coronary Heart Disease Risk Factors
Authors: Oh Eui Geum , Bang So Yeon , Hyun Sa Saeng , Kim Soo Hyun , Jeon Yong Kwan , Im Jee Aee , Lee Mi Kyung , Lee Jung Eun , Chu Sang Hui
Published: July 2021

Purpose
: This study was to examine the effects of a six-month therapeutic lifestyle modification (TLM) program on metabolic coronary heart disease risk parameters in post menopausal Korean women with metabolic syndrome (Mets).

Methods:
A randomized controlled design was used. Fifty four women with Mets were randomly assigned to receive a TLM program. The program was a six-month supervised weekly TLM session consisted of health monitoring, education, aerobic exercise, and a low calorie diet with low carbohydrate (≈1300Kcal/d). Those in the control group received a booklet about MetS and were instructed to maintain their usual diet and activities. Anthropometric measures, blood pressure, and serum cardio metabolic parameters were measured at before, during (month 3), completion (month 6), and after completion of the TLM program (month 12). Mixed-model repeated measures ANCOVA with control of age was used.

Results:
Statistically significant treatment-by-assessment time interaction was observed for waist circumference (
P
= .010). Waist circumference decreased in the TLM group (
d
= − 9cm, 9.5%) over 12 months, compared with standard recommendation group (
d
= − 2cm). Systolic blood pressure (
d
= − 5mmHg,
P
= .017), HDL-cholesterol (
d = +
4.6mg/dl,
P
= .041), and Triglyceride (
d
= + 3 mg/dl,
P <
.001) showed both treatment and time main effects. BMI showed a significant treatment effect (
P <
.001) and LDL-C had a time effect (
P
= .011). Fasting blood sugar (FBS) did not show treatment by time interaction or main effects (
P >
.05), however, treatment group returned to the normal range of FBS at month 12, while it did not in control group.

Conclusion
: The results indicate that 6-month systematic TLM program have considerable beneficial effects on cardiometabolic risk factors in women with MetS.
Abstract 2558: Vascular Healing Following Comparator Drug-Eluting Stents Implantation in an Atherosclerotic Rabbit Iliac Model - A Model of More Predictive in Human Responses to Drug-Eluting Stents
Authors: Nakazawa Gaku , Wilcox Josiah N , Melder Robert , Pruitt Sean , Kolodgie Frank D , Virmani Renu
Published: July 2021

Drug eluting stents (DES) induce delayed arterial healing in man, however, the ideal animal model for predicting the vascular response to DES placement in man is lacking. Hypercholesterolemic (1% cholesterol diet for 5 weeks followed by 0.025% for 4 weeks before stent implantation) New Zealand White rabbits (n=21) received bilateral stents implanted in iliac arteries. Animals were randomized to bare metal stent (Driver), zotarolimus- (Endeavor), sirolimus- (Cypher), and everolimus- (Xience V) eluting stents. Stents were harvested 4 weeks after stent implantation. Morphometric/pathologic analysis was performed. Macrophage infiltration was assessed by RAM-11 immunostaining. Blood cholesterol levels peaked at 5 weeks after initiation of the cholesterol diet which ranged from 1400 –2000mg/dL, followed by ideal range of 500 to 800mg/dL at 9 weeks (stent implantation), and were similar among groups. While EEL, and stent area were comparable in all groups, neointimal formation was significantly more suppressed in Xience and Cypher followed by Endeavor. All DES showed significantly less neointimal formation versus Driver by suppressing cell proliferation (reduced BrdU positive cells, in intima and media). Fibrin deposition, the prevalence of uncovered struts, and the number of adherent luminal inflammatory cells were greater in Xience and Cypher as compared to Driver and Endeavor. All DES showed significantly less macrophage infiltration as compared to BMS. An atherosclerotic rabbit model mimics neointimal formation in various DES and BMS similar to man with greater number of uncovered stent struts and luminal inflammation seen in Cypher and Xience as compared to Endeavor and Driver. Therefore, the atherosclerotic model is a more appropriate model for the assessment of DES efficacy as well as safety. Morphometric Analysis