bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 901: Effects of Bucindolol on Cardiovascular Mortality and Morbidity are Determined by the Beta-1 389 Arg/Gly Receptor Polymorphism
Authors: White Michel , Carson Peter , Anand Inder S , Gottlieb Stephen S , Lindenfeld JoAnn , O’Connor Christopher , Miller Alan , Plehn Jonathan , Silver Marc A , Fiuzat Mona , Robertson Alastair D , Bristow Michael R
Published: July 2021

Introduction:
Bucindolol is a nonselective beta-adrenergic blocker with potent sympatholytic properties. The Beta-blocker Evaluation of Survival Trial (BEST) reported that the administration of bucindolol resulted in a nonsignificant decrease in total mortality (HR = 0.89 (0.78, 1.02), unadjusted p=0.10) in patients with advanced, NYHA Class III-IV heart failure (HF). Recent observations from that trial also reported that the amino acid arginine (Arg/Arg) or glycine (any Gly) in position 389 of the beta-1 receptor plays a significant role on the clinical response to bucindolol. The impact of bucindolol on cardiovascular mortality and morbidity (cardiovascular hospitalizations) has been incompletely investigated, because hospitalizations had been evaluated from case report forms (CRFs) only, and never adjudicated by the endpoints committee (EPC).

Methods:
The BEST data base consists of 2708 patients with a mean follow-up of 2.0 years. Cardiovascular (CV) mortality and hospitalizations have now been evaluated by EPC, which further subclassified total hospitalizations into cardiovascular (CV) and those due to worsening heart failure (HF). The impacts of Arg or Gly encoded at amino acid position 389 on endpoints were further investigated in the 1040 patient substudy.

Results:
Time to event results for adjudicated CV endpoints are presented below.

Conclusions:
Chronic administration of bucindolol results in a significant reduction in cardiovascular hospitalizations and mortality. Effects on either are strikingly beta-1 389 Arg/Gly specific, with the higher functioning, Arg/Arg version of the receptor associated with large treatment effects and Gly carriers exhibiting little or no evidence of efficacy. Genetic targeting of the β
1
-ΑR 389 polymorphism may improve the clinical responses to bucindolol for CV mortality and morbidity.
Abstract 905: Alternative Splicing of
HMGCR
is Associated with Plasma LDL Cholesterol Response to Simvastatin
Authors: Medina Marisa W , Gao Feng , Ruan Weiming , Crawford Dana , Rotter Jerome I , Krauss Ronald M
Published: July 2021

Statins can substantially lower plasma LDL cholesterol and reduce risk for coronary heart disease, but their efficacy varies among individuals. To test whether this variation is related to single nucleotide polymorphisms (SNPs) in the gene for 3-hydroxy-3-methylglutaryl-coenzyme A reductase (
HMGCR
), the key regulatory enzyme of cholesterol synthesis and the target of statin inhibition, we resequenced
HMGCR
exons, splice sites and 1.5kb of 5′ and 3′ flanking regions in 96 individuals. Subjects were equally derived from the upper and the lower 5% of the LDL-response distribution from the Cholesterol and Pharmacogenetics (CAP) study, a 6 week trial in which 944 subjects were treated with 40 mg/day simvastatin. Intronic SNP 20144 (rs3846662) was significantly (p<0.05) associated with high versus low LDL cholesterol response status, and was part of a previously reported haplotype associated with a 14.8% smaller reduction in LDL cholesterol in the CAP cohort. Since SNP 20144 is adjacent to exon 13, a site of
HMGCR
alternative splicing, we measured expression levels of the splice variant lacking exon 13 (
HMGCRv 1
) in 170 immortalized lymphocytes derived from the CAP subjects, and found that the SNP was associated with greater statin-induced
HMGCR
alternative splicing (p<0.05). Moreover, there was a significant correlation between the increased magnitude of expression of
HMGCRv 1
following statin exposure
in vitro
and smaller reductions of total cholesterol, LDL cholesterol, apoB and triglycerides in the plasma of the corresponding subjects
in vivo
(p<0.0001). There was no relationship between the full-length transcript and
in vivo
response. Additionally, we used siRNA to specifically knock-down expression of the full-length
HMGCR
transcript by 68%, resulting in cells enriched in the
HMGCRv 1
transcript. We then measured HMGCR enzyme activity and found that the remaining enzyme demonstrated 9 –15% greater residual activity in the presence of simvastatin concentrations ranging from 0.15nM to 6.0nM (n=10). These results suggest that
HMGCR
genetic variation influences the production of an HMGCR isoform with reduced statin sensitivity, and that variation in expression of this isoform is a determinant of inter-individual differences in statin response.
Abstract 911: Lacking SOCS3 Gene in Macrophages Prevents the Development of Atherosclerosis in ApoE Deficient Mice
Authors: Yamamoto Tomoka , Yasukawa Hideo , Koga Mitsuhisa , Sugi Yusuke , Fukui Daisuke , Futamata Nobuyoshi , Tahara Nobuhiro , Kai Hisashi , Imaizumi Tsutomu
Published: July 2021

Background
: Atherosclerosis is now generally accepted as a chronic inflammatory disease characterized by accumulation of blood-derived macrophages in the arterial wall. It has been shown that suppressor of cytokine signaling 3 (SOCS3) is an intrinsic negative regulator of interleukin-6 (IL-6)-STAT3 signaling that plays an essential role in inflammatory response. In the present study, we determined whether STAT3-mediated IL-6 signaling and SOCS3 within the macrophages would play an important role in the development of atherosclerosis.

Methods and Results
: We examined the activation of STAT3 and expressions of IL-6 family cytokines and SOCS3 in the aorta of apolipoprotein E knockout mice (ApoE-KO) and control mice by western blot, immunohistochemical staining and RT-PCR. Western blot revealed that STAT3 is phosphorylated in the atherosclerotic lesions from ApoE-KO mice but not in those from control mice. Dual immunohistochemical staining revealed that phosphorylated STAT3 and macrophage marker, MOMA2, were co-expressed in the atherosclerotic plaque in ApoE-KO deficient mice, suggesting that the STAT3 signaling pathway within the macrophages was activated in atherosclerosis. Next, to investigate the role of STAT3-mediated IL-6 signaling and SOCS3 in macrophages during atherogenesis, we created ApoE-KO mice with a macrophage-restricted deletion of SOCS3 gene (ApoE/SOCS3-KO). We found that atherosclerotic lesion area by oil-red O staining was significantly reduced in ApoE/SOCS3-KO (n=17) compared with control ApoE-KO mice (n=13) (4.63±0.43 vs. 8.29±0.50 %, p<0.01). In in vitro analyses, enzyme-linked immunosorbent assay revealed that LPS-induced TNF-alpha production were significantly smaller in SOCS3 deficient macrophages compared with wild type macrophages (n=8, 2238±155 vs. 992±83 pg/ml, p<0.01). Also we found that IL-6-induced oxidized-LDL uptake was 42% reduced in SOCS3 deficient macrophages compared with wild type macrophages (p<0.01).

Conclusion:
Our data show that the deletion of SOCS3 in macrophages prevents the development of atherosclerosis in mice, possibly by inhibiting the inflammatory cytokine production and oxidized-LDL uptake in macrophages.
Abstract 912: Spleen Tyrosine Kinase Regulates TLR4 dependent Macropinocytosis And Lipid Accumulation In Macrophages
Authors: Choi Soo-Ho , Almazan Felicidad , Miller Yury I
Published: July 2021

Toll-like receptors play a key role in the signaling pathways of innate immune response and have been recently shown to contribute to vascular inflammation and atherosclerosis. Oxidation of LDL is considered a leading mechanism of atherogenesis, and in our previous studies, we demonstrated that minimally modified LDL (mmLDL) induced TLR4-dependent chemokine secretion as well as robust actin polymerization and spreading of macrophages. We noticed that these mmLDL-induced cytoskeletal rearrangements led to extensive vacuolization, characteristic of macropinocytosis. In this study, we examined the mechanisms of TLR4-induced actin polymerization and macropinocytosis. It has been reported that spleen tyrosine kinase (Syk) regulates actin cytoskeleton and that it may interact with TLR4. We found in the immunoprecipitation experiments that mmLDL induced Syk association with TLR4 as well as Syk phosphorylation. Next we developed a J774 macrophage cell line, which stably expressed Syk shRNA and had the Syk expression reduced by 70% or more. Using this Syk-deficient macrophage cell line and specific Syk inhibitors, we demonstrated that Syk is necessary for mmLDL-induced activation of Ras and phosphorylation of Raf-MEK1-ERK1/2. The ERK1/2 activation resulted in phosphorylation of paxillin and an increase in the formation of N-WASP/Arp2 complex, which initiates actin polymerization. Taken together, these results suggest that Syk is a critical kinase in the mmLDL macrophage activation and that the mmLDL-induced cytoskeleton signaling. In addition, we demonstrated that mmLDL induced macrophage uptake of native LDL and of fluorescent 10 KDa dextran, and that this effect was Syk-dependent. These data suggest a novel pathway for the mmLDL-induced macropinocytosis in macrophages, which may constitute a mechanism for macrophage lipid accumulation and foam cell formation, a characteristic step in the development of atherosclerosis.
Abstract 927: The Role Of Peroxiredoxins As Mechanosensitive Antioxidants In Endothelial Cells
Authors: Mowbray Amy , Kang Sang Won , Rhee Sue Goo , Jo Hanjoong
Published: July 2021

Atherosclerosis is an inflammatory disease occurring primarily in curved or branching regions of the arterial tree where disturbed flow patterns, such as oscillation, exist. We have previously shown that oscillatory shear stress (OS) increases reactive oxygen species (ROS) levels in endothelial cells, while laminar shear stress (LS) reduces ROS compared to static controls. OS stimulation of ROS has been shown to occur in an NADPH oxidase-dependent manner. However, the mechanism by which LS removes ROS remains unclear. Peroxiredoxins (PRX) are a family of antioxidant proteins that have been linked to the prevention of oxidative stress and inflammation, but their role in atherosclerosis is unknown. Here, we hypothesize that shear stress regulates ROS levels in endothelial cells by controlling antioxidant peroxiredoxins. To test this hypothesis, bovine aortic endothelial cells (BAEC) were subjected to static, laminar, and oscillatory fluid flow conditions via cone-and-plate viscometer. Western blot analysis and immunofluorescent staining were used to evaluate the expression and subcellular localization of six known mammalian peroxiredoxins (PRX I-VI). Immunoblots indicated that BAEC express all six isoforms of peroxiredoxin proteins and that LS upregulated PRX I levels significantly compared to static controls and OS. Immunofluorescence also showed a distinct subcellular localization of each PRX: PRX I, II, IV, V and VI in the cytoplasm, PRX I, IV and V in the Golgi, PRX III in the mitochondria, and PRX I in the nucleus. These results indicate that peroxiredoxins are mechanosensitive antioxidants, removing ROS in a subcellular-specific manner. Based on these data, we suggest that peroxiredoxin antioxidants are likely involved in the molecular mechanisms that control shear stress-dependent atherosclerotic plaque development.
Abstract 947: Dpp10 - A New Putative Regulatory β-subunit Of I
to
In Failing And Non-failing Human Heart
Authors: Radicke Susanne , Cotella Diego , Bortoluzzi Alessio , Ravens Ursula , Sblattero Daniele , Wettwer Erich
Published: July 2021

Background:
Recently we reported that the dipeptidyl-aminopeptidase-like protein DPP6 serves as a regulatory β-subunit for cardiac Kv4.3 channels. DPP6 is up-regulated in human failing hearts in which Kv4.3 and the β-subunit KChIP2 are down-regulated. Here, we provide evidence for the presence of a new member of this protein family, i.e. DPP10, in failing and non-failing human hearts and investigate the role of this putative β-subunit in regulating transient outward current.

Methods:
mRNA was extracted from samples of 5 failing and 5 non-failing human hearts and quantified by realtime PCR. Functional interaction of DPP10 and Kv4.3 was studied in co-expression experiments (Chinese hamster ovary CHO cells) with standard voltage clamp techniques.

Results:
Expression level of DPP10 was 85±13 fg/ng in failing and 33±6 fg/ng total RNA in non-failing hearts (P<0.01), amounting to a 2.6fold reduction. In comparison, DPP6 was up-regulated 1.3fold (P<0.001). After co-expression of Kv4.3 and DPP10 in CHO cells, channel complexes were verified in the plasma membrane by immunostaining, suggesting proper trafficking as with co-expression of Kv4.3 and KChIP2. Expression of Kv4.3 alone failed to yield I
to
current, but robust I
to
amplitudes were measured after co-expression of Kv4.3 and DPP10, DDP6, or KChIP2. Compared with the conventional co-expression combination of Kv4.3 and KChIP2, kinetics of I
to
inactivation were accelerated in Kv4.3 plus DPP10 channels (τ
fast
: 56± 3 ms and 5.9±0.4 ms, respectively; P<0.01), however, recovery from inactivation was not affected (τ
rec
: 53± 7 ms vs. 58±13 ms). Co-expression of Kv4.3 with KChIP2 plus DPP10 maintained rapid inactivation of I
to
(τ
fast
8±1ms), but enhanced recovery kinetics (τ
rec
: 13±2 ms, P<0.001). The role of glycosylation for channel kinetics was studied with the glycosylation inhibitor tunicamicin (10 μg/ml; 24h). In the presence of tunicamicin, I
to
inactivation and recovery were slowed (τ
fast
51± 4 ms and τ
rec
: 42±3 ms), suggesting importance of glycosylation for channel function. Kinetics were similarly slowed when extracellular domain of DDP10 was deleted (τ
fast
: 48±5 ms, τ
rec
: 30± 4 ms).

Conclusion:
DPP10, like DPP6 and KChIP2, contributes to regulation of I
to
in normal and diseased human hearts.
Abstract 957: A Novel Interaction of Cardiac Troponin T with PKA Regulatory Subunits I and II
Authors: Sumandea C. Amelia , GarcÃa-CazarÃn Mary L , Bozio Catherine H , Balke C. William , Solaro R. John , Sumandea Marius P.
Published: July 2021

Increasing evidence suggests that cardiac myofilament activity is modulated by posttransla-tional modifications of cardiac troponin (cTn). In an attempt to identify novel proteins that associate with and modify cTn, we used the full-length human cardiac TnT as bait in the yeast two-hybrid system, and a human heart cDNA library as prey. Our screen of > 2×10
6
cDNA clones with hcTnT as bait, identified >1000 clones that grew on high-stringency plates, and 210 that turned the cells blue in the presence of α-gal, indicating positive interactions with hcTnT. DNA sequencing of the 210 clones led to the identification of 10 gene products: cTnI (identified in 180 clones), PKA-RIα, MLC2v, Dystrophin, PDZ and LIM domain 5, CGI-94, Muscle Creatine Kinase, ANF, Mitofusin-2 and an uncharacterized gene. HA-tagged clones (above) and myc-tagged cTnT were individually expressed using a rabbit reticulocyte lysate system and immunoprecipitated (IP) by agarose conjugated anti-myc and anti-HA antibodies. Therefore, the interaction of these proteins with cTnT was verified in a mammalian cellular milieu. Our finding suggests that cTnT, through its interaction with PKA regulatory domains (R), anchors PKA to the myofilament in close proximity to cTnI. This would explain the rapid, PKA dependent, phosphorylation of cTnI during increase intracellular cAMP levels following beta-adrenergic stimulation. While, PKA type I holoenzyme is predominantly cytoplasmic, the majority of PKA type II associates with cytoskeletal structures. Binding of PKA-RI and PKA-RII to intact and truncated forms of cTnT (amino acids 1–117; 118 –288; 1–180; 181–288) were determined by yeast two-hybrid and by IP assays. Our results demonstrate, for the first time, that both PKA-RI and RII associate with cTnT, and PKA-RI binds to a specific region of cTnT-1–180 fragment, whereas PKA-RII binds to a region of cTnT-181–288 fragment. PKA-dependent phosphorylation of cTnI-Ser 23/24 has profound effects on cardiac dynamics: modulating cross-bridge kinetics, energy consumption and calcium sensitivity. Therefore, it is important to have a mechanistic understanding of events that mediate and affect PKA-dependent cTnI phosphorylation.
Abstract 964: Effect of Volume Overload on Left Ventricular Torsion and Extra Cellular Matrix
Authors: Nagaraj Hosakote M , Denney Thomas S , Lloyd Steven G , Calhoun David , Aban Inmaculada , Corros Cecilia , Zoghbi Gilbert J , Perry Gilbert J , Dell’Italia Louis J , Gupta Himanshu
Published: July 2021

Background:
Muscle fibers are arranged in a spiral network and are connected by extracellular matrix (ECM). LV torsion is increased in the pressure overloaded heart where there is an increase in ECM. However, torsion and its relation to ECM have not been systematically studied in the volume overloaded heart.

Hypothesis:
The volume overloaded heart has a decrease in LV torsion due a loss of ECM.

Methods:
Primary mitral regurgitation (MR) (n=29), resistant hypertension (HTN) (n=77) and normal volunteers (NL) (n±37) were studied. Comprehensive cardiac magnetic resonance imaging (MRI) with tissue tagging was performed and analyzed using three-dimensional data set. Torsion was computed by fitting a B-spline deformation model in prolate-spheroidal coordinates to the tag line data. A subset of MR subjects had LV collagen assessed by picric acid Sirius red from biopsy samples taken at the time of surgery.

Results:
LV ejection fraction was 65% in MR and 70% in HTN. MR demonstrated eccentric remodeling and HTN demonstrated concentric remodeling. HTN had significantly higher torsion angle and systolic twist compared to NL and MR. This was associated with a simultaneous decrease in longitudinal strain. In contrast, MR patients had similar torsion indices, circumferential and longitudinal strains compared to NL. LV biopsy in MR demonstrated a decrease in interstitial collagen compared to NL.

Conclusions:
As opposed to the pure volume overloaded heart, LV torsional forces are increased in the pressure overloaded heart. This difference may be related to a rearrangement of the laminar structure due to a differential effect on ECM in the volume overloaded versus the pressure overloaded heart.
Abstract 976: Hyperpolarized Mitochondria in Right Ventricular Hypertrophy: a Target for Ventricular-Specific Therapies
Authors: Nagendran Jayan , Gurtu Vikram , Dyck Jason R , Ross David B , Rebeyka Ivan M , Michelakis Evangelos D
Published: July 2021

Objectives
: The right ventricle (RV) fails quickly after increases in its afterload (pulmonary hypertension) compared to the LV (systemic hypertension). We hypothesized that the suboptimal performance of the hypertrophied RV (RVH) is caused by metabolic adaptation during hypertrophy. Mitochondria regulate metabolism, and mitochondrial membrane potential (ΔΨm) is a surrogate for mitochondrial function.

Methods/Results
: We measured ΔΨm in normal and hypertrophied myocardial specimens from 11 patients and in a rat model of RVH, using Tetramethylrhodamine methyl ester, a positively-charged dye that selectively localizes within the negatively charged mitochondria. ΔΨm was significantly higher in the normal LV compared to normal RV, and was the highest in RVH, both at the myocardium and isolated cardiomyocyte levels (p<0.01 for both). The increase in ΔΨm was recapitulated in an in vitro model of hypertrophy, where isolated neonatal cardiomyocytes were exposed to phenylephrine. This increase in ΔΨm was reversed by both a direct inhibitor of NFAT (Nuclear-Factor-of-Activated-T-lymphocytes, a transcription factor that regulates cardiac metabolism in development and hypertrophy) and Dichloroacetate (DCA, a metabolic modulator that has been shown to depolarize mitochondria and inhibit NFAT, reversing the phenotype of disease in both cancer and pulmonary hypertension) (p<0.01 for both). In ex-vivo Langendorff perfusions DCA acutely increased RV inotropy in rats with RVH (p<0.01) but not normal RVs, suggesting that the increased ΔΨm in RVH might be associated with its suboptimal performance in patients with pulmonary hypertension.

Conclusions
: The significant differences in ΔΨm between RV, LV, and RVH, can be targeted metabolically by DCA to improve contractility in RVH and provide a framework for development of novel RV-specific therapies.
Abstract 978: Novel Endothelial-protective Effect of High Density Lipoprotein: Down-regulation of Toll-like Receptor 4
Authors: Spillmann Frank , Van Linthout Sophie , Subasigüller Aysun , Bücker-Gärtner Carola , Meloni Meloni , Egorova Marina , De Geest Bart , Schultheiss Heinz-Peter , Tschöpe Carsten
Published: July 2021

Introduction:
Endothelium-derived Toll-like receptor (TLR) 4 is known to be the key molecule in lipopolysaccharide (LPS)-induced neutrophil sequestration into lungs. The aim of our study was to investigate whether the endothelial-protective effects of HDL also include regulation of Toll-like receptor (TLR) 4. Therefore, we analyzed the effect of HDL supplementation on TLR4 expression in human aortic endothelial cells (HAEC) and the effect of increased HDL following adenoviral apo AI (
Ad.AI)
gene transfer (GT) on lung TLR4 expression in an experimental model of LPS-induced endotoxic shock.

Methods:
HDL (50 μg/ml) was supplemented to HAEC in the presence/absence of LPS (100 ng/ml). Surface-TLR4 expression on HAEC was analyzed by FACS. C57BL/6 mice were i.v. injected with 5 x 10
10
total particles of
Ad.AI
or with
Ad.Null
. Fourteen days hereafter, mice were i.p. injected with LPS (80 mg/kg) or with saline and 20 hours afterwards sacrificed. For survival studies, endotoxic shock was induced in 25 mice per group. Lung myeloperoxidase activity (MPO), as a marker of neutrophil infiltration was analysed. Lung TLR4-mRNA expression was determined by real-time PCR, plasma interferon-gamma (INFγ) levels by ELISA.

Results:
HDL supplementation significantly reduced TLR4 expression on HAEC.
Ad.AI
resulted in human apo AI expression levels of 80 ± 10 mg/dl at day 14. By 24 hours after LPS injection, 81% of LPS-treated
Ad.Null
and saline mice died, in contrast to 50% of
Ad.AI
LPS-treated mice. This was associated with a 1.6- and 1.7-fold (p<0.05) lower lung/body weight ratio in
Ad.AI
compared to
Ad.Null
and saline LPS-injected mice. LPS-induced lung TLR4 mRNA expression was 2.0-fold (p<0.05) reduced after human apo AI-GT compared to LPS controls. Moreover, human apo AI-GT significantly reduced neutrophil infiltration in LPS-treated mice, as indicated by 1.8-fold (p<0.05) and 1.9-fold (p<0.05) reduced MPO activity compared to controls, respectively. Plasma INFγ-levels were 2.8-fold and 2.2-fold (p<0.05) lower versus LPS-treated
Ad.Null
and saline mice, respectively.

Conclusion:
The HDL-mediated reduction in lung TLR4 expression contributes for the reduced neutrophil infiltration and subsequently may account for improved mortality rate.
Abstract 981: Hypoxia Results in Exacerbation of Pulmonary Arterial Hypertension (PAH) in Male Mice with Deletion of PPARγin Smooth Muscle Cells (SMC) but Females are Protected by Adiponectin Mediated Induction of PPARα
Authors: Rubinos Laura H , Hansmann Georg , El-Bizri Nesrine , Guignabert Christophe , De Jesus Perez Vinicio A , Rabinovitch Marlene
Published: July 2021

Lung tissues of patients with PAH have reduced levels of the peroxisome proliferator activated receptor gamma (PPARγ) and we previously reported that transgenic male mice with deletion of PPARγ in arterial smooth muscle (SM22CrePPARγ
flox/flox
) develop spontaneous PAH. Moreover, PPARγ is necessary in mediating bone morphogenetic protein (BMP-2) inhibition of PDGF-BB induced SMC proliferation. We therefore
hypothesized
that PAH would be exacerbated in SM22CrePPARγ
flox/flox
mice under conditions like chronic hypoxia (3 weeks, FiO
2
=0.10) that induce PDGF-BB mediated SMC proliferation.

Results:
Consistent with this hypothesis, we report a more severe PAH phenotype only in the male SM22CrePPARγ
flox/flox
mice, as measured by greater right ventricular systolic pressure (RVSP: 38.6 vs. 30.9 mmHg), right ventricular hypertrophy (RVH=right ventricle/left ventricle+septum: 0.58 vs 0.35), and arterial muscularization (90.6 vs 60.3 %), when compared to littermate controls (p<0.05, n=6 – 8 per group, for all comparisons). Interestingly, female SM22CrePPARγ
flox/flox
mice were resistant to spontaneous or chronic hypoxia-induced PAH (RVSP: 18.5mmHg). We investigated the acute hypoxic vasoconstrictive response and found it exaggerated in the male but suppressed in the female (RVSP: 43.12 vs 20.42mmHg) SM22CrePPARγ
flox/flox
mice. We found no reduction in endothelin-1 (P<0.05) in the female vs. male SM22CrePPARγ
flox/flox
mice but serum levels of adiponectin were twofold higher (14.5 vs 6.9 μg/ml; p<0. 001) in females under normoxia and hypoxia. We further show through cell counts, that in pulmonary artery (PA) SMC from male SM22CrePPARγ
flox/flox
mice, BMP2 fails to suppress PA SMC proliferation in response to PDGF-BB (p<0.01; n=3) but exogenous adiponectin can ‘rescue’ this phenotype (p<0.01, n=3). The mechanism appears to be related to adiponectin mediated induction of PPAR alpha (PPARγ mRNA relative level: 0.37 vs 0.42 after adiponectin pretreatment, p<0.01), which we report for the first time in PA SMC.

Conclusion
: These findings strengthen the potential efficacy of PPAR-α/ γ agonists in the treatment of PAH patients with low endogenous levels of PPAR-γ and/or adiponectin, such as those with BMP-RII dysfunction or insulin resistance.
Abstract 997: Innate Immune Adaptor MyD88 Mediates Myocardial Inflammation And Infarction In A Mouse Model Of Ischemia-reperfusion Injury
Authors: Zhao Huailong , Xu Xinhua , Schmidt Ulrich , Chao Wei
Published: July 2021

INTRODUCTION: The innate immune response to ischemia-reperfusion (I/R) is, by far, the most common cause of myocardial inflammation and contributes to ischemic myocardial injury. MyD88 is an adaptor protein critical for transducing signals from most innate immune receptors and plays an important role in host defense against microbial infection; but its role in non-infectious myocardial inflammation and injury following I/R is unclear.

METHODS: I/R injury was studied in wild-type (WT) and MyD88-deficient (MyD88−/−) mice. Mice were anesthetized and mechanically ventilated. The heart was exposed via intercostal thoracotomy. The left coronary artery was ligated for 30 min followed by reperfusion. The coronary artery was re-ligated at 24 hours and fluorescent microsphere was injected into LV to detect area-at-risk (AAR). The hearts were harvested, sectioned, and examined for inflammation or infarct size. Myocardial neutrophil infiltration [GR-1 immunohistochemistry and myeloperoxidase activity] and NF-kB signaling (I-kBa degradation and NF-kB binding activity) were examined to assess myocardial inflammation. In ex vivo studies, isolated mouse hearts were exposed to global ischemia for 20 min followed by reperfusion for 30 min.

RESULTS: In vivo I/R induced significant neutrophil infiltration and NF-kB activation in myocardium in WT mice compared with the sham-operated mice. However, in MyD88−/− mice subjected to the same I/R, the level of neutrophil recruitment and NF-kB signaling was significantly lower compared with WT mice. The ratio of AAR to LV was similar in WT and MyD88−/− mice (34 +/− 3% vs. 41 +/− 3%). However, the ratio of MI to AAR was 58% less in MyD88−/− than in WT mice (14 +/− 2% vs. 33 +/− 6% (p < 0.014, n = 11–12). In a Langendorff model of I/R injury, a system devoid of circulating blood components, global I/R induced similar MI sizes, as a fraction of LV size (MI/LV), in MyD88−/− and WT mice (34 +/− 6% and 25 +/− 7%, respectively, p = 0.3, n = 8).

CONCLUSIONS: These studies demonstrate that systemic MyD88 deficiency decreases myocardial inflammation and infarction after ischemia-reperfusion in vivo and support the hypothesis that MyD88 mediates ischemic myocardial injury via mechanisms that are dependent upon circulating blood components.
Abstract 123: Role of Valsartan (V) Alone or in Combination with Simvastatin (S) in Reducing Inflammation of Thin Cap Fibroatheromas
Authors: Chatzizisis Yiannis S , Jonas Michael , Coskun Ahmet U , Beigel Roy , Stone Benjamin V , Maynard Charles , Gerrity Ross G , Daley William , Rogers Campbell , Edelman Elazer R , Feldman Charles L , Stone Peter H
Published: July 2021

Objectives:
We investigated the role of V alone or in combination with S on the histomorphologic characteristics of thin cap fibroatheromas (TCFAs), and tested the hypothesis that V or VS attenuate the proinflammatory effect of low endothelial shear stress (ESS). We used vascular profiling of swine coronary arteries for in vivo assessment of ESS to prospectively identify these low ESS areas that are prone to develop TCFAs.

Methods:
12 diabetic hyperlipidemic swine were allocated into 3 treatment groups: placebo (P, n=4), V (n=4) and VS (n=4). Blood pressure, serum cholesterol and glucose were similar between the treatment groups. IVUS-based geometrically correct 3D reconstruction of the coronary arteries was performed at baseline (wk 23) and follow up (wk 30). Baseline ESS was calculated using computational fluid dynamics and plaque-free subsegments of interest were identified (n=109). Coronary arteries (n=31) were harvested at follow up, cryosectioned at the subsegments of interest and stained histologically. Intima/media ratio and inflammation (CD45) were quantified. Lesions were classified into atheromas without evidence of fibrous cap (n=82) and TCFAs (n=60).

Results:
V alone or in combination with S reduced the amount of plaque inflammation, particularly in TCFAs (Fig A

). V and VS attenuated the proinflammatory effect of local low ESS compared to P (Fig B

).

Conclusion:
V alone or in combination with S exerts a stabilizing effect of reducing plaque inflammation, even in high-risk regions with low ESS. These results suggest a mechanism of regional atheroprotection associated with V or VS.
Abstract 126: Chlamydia Pneumoniae Induces Foam Cell Formation Of Macrophages By Activating The TLR4/TRIF/IRF3 Pathway
Authors: Chen Shuang , Sorrentino Rosalinda , Shimada Kenichi , Crother Timothy , Arditi Moshe
Published: July 2021

Background:
Chlamydia pneumoniae
(CP) induces macrophage foam cell formation (FCF), a key event in early atherosclerosis, in the presence of low-density lipoprotein (LDL). Recent studies have indicated the role of Toll-Like Receptors in atherogenesis. Liver X receptors (LXR) are nuclear receptors that play central roles in the transcriptional control of lipid metabolism and determinants of atherosclerosis. Induction of LXR-activated genes has also been shown to influence the pathogen pattern recognition activity of the Toll-like receptors 3 and 4 (TLR3/4). The TLR and the LXR pathways converge on the transcription factor IRF3.

Objective:
We hypothesized that TLR and the LXR and IRF3 pathways participate in CP infection mediated FCF and acceleration of atherosclerosis, and that the MyD88- independent pathway via TLR4/TRIF and IRF3 play a role in this acceleration.

Methods:
Peritoneal macrophages were isolated from C57BL/6 wild type (WT) mice, IRF3
−/−
mice, TLR4
−/−
mice and TRIF
−/−
mice. Cells were treated with UV killed CP (UVCP, 5x10
5
IFU) with or without ox-LDL (100 μg/ml) in the presence or absence of LXR agonist GW3965 (2nM). LPS (10 ng/ml) and PolyI:C (1μg/ml) were used as positive controls as TLR4 and TLR3 ligands, respectively. FCF was examined by Oil Red O staining. The percentages of foam cells in total macrophages were quantified.

Results
: FCF was significantly reduced in IRF3−/− cells compared with WT cells stimulated with UVCP plus ox-LDL. Foam cells induced by LPS with ox-LDL were also significantly reduced in IRF3−/− cells compared to WT cells (p<0.05). Furthermore, the synthetic LXR agonist GW3965 significantly diminished CP induced FCF in WT cells. FCF was significantly reduced in TLR4−/− and TRIF−/− macrophages compared to WT cells when stimulated with UVCP with ox-LDL (p<0.05).

Conclusion
:
Chlamydia pneumoniae
infection can activate the TLR4/TRIF/IRF3 pathway and does play an important role in CP- mediated foam cell formation in macrophages. Therefore, infections such as the one caused by CP, can trigger the TLR4/TRIF/IRF3 pathway leading to the down regulation of LXRs and shifting of cholesterol transport toward pro-foam cell production and thereby accelerating atherogenesis..

Supported by NIH grants AI 067995 and HL66436 to MA.
Abstract 1017: Post-translational Modification Of GATA-4 Involved In The Differentiation Of Monkey ES Cell Into Cardiac Myocytes
Authors: hosseinkhani mohsen , Hosseinkhani Hossein , Khademhosseini Ali
Published: July 2021

Transplantation of embryonic stem (ES) cells into infracted myocardium has been shown to preserve left ventricular function in rodents. Before application of ES cell therapy in humans, however, it is critical to perform pre-clinical studies in large animals such as primates. Characteristics of cynomolgus monkey ES cells are similar with those of human ES cells, but quite different from those of mouse ES cells. Differentiation of Embryonic stem (ES) cells into cardiac myocytes requires activation of a cardiac-specific gene program. Histone acetytrans-ferases (HATs) and Histone deactylases (HDACs) govern gene expression patterns by being recruited to the target genes through association with specific transcription factors. One of the HATs, p300, serves as a coactivator of cardiac-specific transcription factors such as GATA-4. The HAT activity of p300 is required for actylation and DNA binding of GATA-4 and its full transcription activity as well as for promotion of a transcriptionally active chromatin configuration. The role of HATs and HDACs in post-translational modification of GATA-4 during the differentiation of monkey ES cells into cardiac myocytes remained unknown. In an ES cell model of developing embryonic bodies, an acetylated form of GATA-4 and its DNA binding increased concomitantly with the expression of p300 during the differentiation of ES cells into cardiac myocytes. Treatment of ES cells with trichostatin A (TSA), a specific HDAC inhibitor, induced acetylation of histone-3/4 near GATA sites within the atrial natriuretic factor promoter. In addition, TSA augmented the increase in an acetylated form of GATA-4 and its DNA binding during the ES cell differentiation. TSA facilitate the expression of endogenous cardiac β-myosing heavy chain during the differentiation. These findings demonstrate that acetylation of GATA-4 as well as of histone are involved in the differentiation of monkey ES cells into cardiac myocytes.
Abstract 1019: Intracellular Calcium Cycling Mediates Proliferation and Differentiation of Human Cardiac Stem Cells
Authors: Rizzi Roberto , Arcarese Michael L , Esposito Grazia , Bearzi Claudia , Korn Justin A , Di Marco Andrea , Kajstura Tymoteusz J , Hosoda Toru , Steadman Elisabeth , Urbanek Konrad , D’Amario Domenico , Sonnenblick Edmund H , Leri Annarosa , Anversa Piero , Rota Marcello
Published: July 2021

Human cardiac stem cells (hCSCs) are self-renewing, clonogenic and have the ability to differentiate into myocytes, smooth muscle and endothelial cells in vitro and in vivo. Since Ca
2+
plays a crucial role in mechanotransduction and activation of signalling pathways in mature cardiac cells, intracellular Ca
2+
cycling was studied in hCSCs to determine the function of this cation in cell division and commitment to the myocyte lineage. For this purpose, hCSCs were exposed to conditions favouring proliferation and differentiation and affecting intracellular Ca
2+
homeostasis. Moreover, hCSCs were loaded with Fluo-3 and intracellular Ca
2+
levels were monitored by 2-photon microscopy. hCSCs presented spontaneous Ca
2+
spikes mediated by Ca
2+
release from the endoplasmic reticulum (ER). ATP and histamine, which stimulate InsP
3
R-mediated ER Ca
2+
release, increased the occurrence of spikes leading to oscillations in intracellular Ca
2+
. 2-APB, an antagonist of InsP
3
R, inhibited spike formation and oscillatory events. Ryanodine, which acts on the ryanodine receptors, did not alter intracellular Ca
2+
and thapsigargin, a Ca
2+
pump blocker, prevented spontaneous and induced ER Ca
2+
release. Store operated capacitative Ca
2+
entry was evoked by increasing extracellular Ca
2+
after depletion of the ER. Ca
2+
entry was blocked by lanthanum. Additionally, patch-clamp experiments indicated the absence of the voltage-activated L-type Ca
2+
current in hCSCs. Exposure of hCSCs to IGF-1 triggered acutely Ca
2+
spikes and increased chronically their occurrence. Over a period of 24 hours, IGF-1 resulted in more than 100% increase in the proliferation of hCSCs measured by BrdU labelling. Similarly, ATP enhanced proliferation of hCSC by ~60%. Importantly, incubation with 2-APB reduced by ~50% BrdU incorporation and abolished the effect of IGF-1 and ATP on both Ca
2+
spikes and cell proliferation. In the presence of differentiating medium, the frequency of Ca
2+
spikes in active hCSCs increased significantly. Additionally, enhanced Ca
2+
cycling increased the number of hCSCs committed to the myocyte lineage, while attenuations in this phenomenon blunted hCSC differentiation. Thus, InsP
3
R-mediated Ca
2+
spikes play an obligatory role in hCSC growth and differentiation.
Abstract 1039: A Proprotein Convertase Regulates Integrin - MMP Cooperation In VSMCs
Authors: Stawowy Philipp , Meyborg Heike , Baumann Bernadette , Stibenz Dietger , Fleck Eckart , Graf Kristof
Published: July 2021

ECM remodeling is a key feature in atherosclerosis and restenosis. Integrins link the cytoskeleton with the ECM and regulate adhesion/migration. MMPs are the major degrading enzymes important for cell motility. Binding of the MMP-2 zymogen to alpha v beta 3 is necessary for its full activation, thereby coordinating cell motility with membrane-associated proteolysis. The alpha v integrin chain is synthesized as a proprotein that is endoproteolytically activated by furin. Furin is the prototype proprotein convertase, highly expressed in human atherosclerotic lesions. The aim of this study was to investigate the role of furin in integrin -MMP coordination and cooperation in VSMCs.

Methods and Results:
Treatment of VSMCs with either the furin inhibitor dec-CMK (50 umol/L) or an MMP-inhibitor (GM6001; 25 umol/L) significantly inhibited VSMC Matrigel invasion (p<0.05 vs. controls). Immunoblotting demonstrated that dec-CMK inhibited alpha v endoproteolytic activation, but this did not affect alpha v membrane expression assessed by FACS analysis. Zymography revealed that in dec-CMK or TSR1265 (10 umol/L; an inhibitor of MMP-2 binding to alpha v beta 3) treated cells, maturation of the intermediate 68 kDa MMP-2 to its fully active 62 kDa form was significantly decreased (p<0.05 vs. controls). Furthermore, dec-CMK significantly inhibited binding of FITC-conjugated pro-MMP-2 to the cell surface of VSMCs. Immunfluorescence and 3D in gel zymography demonstrated that inhibition of endoproteolytic cleavage of the alpha v integrin inhibits actin rearrangement and focal contact formation upon integrin stimulation (matrix adhesion or PMA treatment), as well as membrane-associated proteolysis.

Conclusion:
Our study demonstrates that endoproteolytic cleavage of alpha v integrin by furin regulates not only integrin activation, but also affects MMP-2 maturation. Endoproteolytic activation of alpha v is required for cytoskeleton rearrangement upon integrin stimulation and focal contact formation, as well as the coordination and cooperation of integrins and MMPs. Therefore furin-convertase may be a novel target in atherosclerosis and restenosis.
Abstract 1078: β-Adrenergic Stimulation of Ca/Calmodulin-Dependent Protein Kinase II (CaMKII) Overexpressing Myocytes Increases Sarcoplasmic Reticulum (SR) Calcium Leak Causing KN-93 Sensitive Arrhythmias
Authors: Wadsack Daniel P , Kohlhaas Michael , Rokita Adam G , Neef Stefan , Maier Lars S
Published: July 2021

CaMKII is associated with hypertrophy, heart failure and alters intracellular Ca homeostasis. An increased SR Ca leak due to phosphorylation of SR Ca release channels by CaMKII leads to decreased SR Ca content and impaired contractility. This loss of Ca from the SR may also contribute to arrhythmias. We investigated whether β-adrenergic stimulation with isoproterenol (ISO) normalizes SR Ca content and whether inhibiting CaMKII reduces arrhythmias. CaMKII-overexpressing rabbit and mouse myocytes were investigated. Cell shortening, Ca fluorescence (fluo-3) and the incidence of arrhythmias were assessed. An arrhythmia-score differentiated between: early-spike-arrhythmias (ESA), late-spike-arrhythmias (LSA) and permanent arrhythmias (PA). ISO (37°C) had significantly different effects on myocytes with acute (24 h, rabbit, n=34) or chronic (22 w, mouse, n=34) CaMKII overexpression vs corresponding control myocytes (LacZ, n=21 or WT n=34). CaMKII overexpression lead to an ISO concentration-dependent (10
−10
-10
−5
mol/L) inotropic but compared to WT (or LacZ, respectively) impaired shortening and Ca transients (two-way ANOVA,
P
<0.05). A similar difference between CaMKII-overexpressing (n=17) and WT (n=19) myocytes was also seen during a shortening-frequency protocol (stepwise increase from 0.1– 4 Hz, two-way ANOVA,
P
<0.05). Arrhythmias spontaneously occurred in CaMKII-overexpressing mouse myocytes. With β-inotropic stimulation (10
−6
mol/L ISO) arrhythmias were increased 6.4-fold. Appearance of ESA and PA could be significantly reduced by KN-93 (1 μmol/L). At a basal stimulation rate of 1 Hz and 10
−7
mol/L ISO, PA could be dramatically reduced by half from control-level 21.43% (KN-92, inactive derivative, n=42) down to 10.87% (KN-93, n=46) arrhythmic events. ESA could be reduced almost 4-fold from 16.67% (KN-92) to 4.35% in the presence of KN-93. We conclude from these data that increasing ISO concentrations exerts positive inotropic effects but cannot normalize altered Ca handling in CaMKII-overexpressing myocytes. This may be due to an increased SR Ca leak under these conditions thus contributing to the arrhythmias observed. CaMKII inhibition clearly can reduce arrhythmias in the presence of β-adrenergic stimulation with ISO.
Abstract 1079: Prevention of Fatal Arrhythmia in a Catecholaminergic Polymorphic Ventricular Tachycardia Mouse Model Carrying Calsequestrin-2 Mutation
Authors: Wakimoto Hiroko , Alcalai Ronny , Song Lei , Arad Michael , Seidman Christine E , Seidman Jonathan G , Berul Charles I
Published: July 2021

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a familial arrhythmia syndrome caused by mutations in the ryanodine receptor (RyR2) or calsequestrin-2 (CASQ2) genes and characterized by exercise or emotional stress-induced sudden death. Beta-adrenergic blockers are only partially effective and other agents have not been widely tested. Recent studies have shown that CPVT is mediated by increased Ca
2+
leak through the RyR2 channel. Our aim was to determine whether agents that inhibit intracellular Ca
2+
leak can effectively prevent CPVT. Methods: The efficacy of intraperitoneal (IP) propranolol (1mcg/g), Mg
2+
(0.002mEq/g), verapamil (8 mcg/g) and diltiazem (8 mcg/g) were tested both in vivo and in vitro using CASQ2 mutant mouse CPVT model. In vivo studies included ambulatory ECG recordings at rest and following epinephrine stress (0.4 mcg/g IP) at baseline and after study drug administration. Experiments for each drug were performed on separate days to avoid confounding effects. In vitro studies included intracellular Ca
2+
transient analysis on isolated cardiomyocytes from mutant mice with and without epinephrine (5.5 μM). Results: All 4 drugs restored sinus rhythm and reduced the frequency of VT episodes induced by epinephrine in CASQ2 mutant mice. Only verapamil completely prevented epinephrine-induced VT in 87% of the mice (p<0.01). Cardiomyocyte studies in basal conditions revealed that Mg
2+
and verapamil inhibited sarcomere contraction and normalized the prolonged Ca
2+
reuptake period in CASQ2 mutants, but did not decrease baseline Ca
2+
peak height. Epinephrine-stressed mutant myocytes had increased diastolic Ca
2+
levels, lower Ca
2+
peak height and spontaneous SR Ca
2+
release events that were partially prevented by verapamil and Mg
2+
. Verapamil was more effective than Mg
2+
in reducing the frequency of spontaneous Ca
2+
releases induced by epinephrine. Conclusions: All 4 agents can inhibit ventricular arrhythmia in CPVT mouse model; however verapamil appears most effective in preventing arrhythmia in vivo and in modifying intracellular abnormal calcium handling. Calcium antagonists might have therapeutic value in CPVT and other RyR2-mediated arrhythmias and should be considered for human clinical studies.
Abstract 1087: Effects of Oral Gap Junction Conduction-Enhancing Antiarrhythmic Peptide GAP-134 on Experimental Atrial Fibrillation in Dogs
Authors: Laurent Gabriel , Leong-Poi Howard , Moe Gordon , Hu Xudong , So Petsy Pui-Sze , Ramadeen Andrew , Doumanovskaia Liia , Konig Andrea , Hennan James , Dorian Paul
Published: July 2021

Background:
Abnormal intercellular communication caused by connexin dysfunction may promote atrial fibrillation (AF).

Objective:
To assess the effect of the gap junction conduction-enhancing antiarrhythmic peptide GAP-134 on AF inducibility and maintenance in a new dog model of atrial cardiomyopathy.

Methods and Results:
Twenty four dogs underwent simultaneous atrioventricular pacing (2 weeks at 220 bpm, atrioventricular delay 0 ms), and were randomly assigned to placebo treatment (PACED-PLACEBO; 12 dogs) or oral GAP-134 (PACED-GAP 134; 12 dogs) (starting at day 0). Percent change in left atrial systolic area (Δ% LASA) from baseline to 2 weeks was calculated using trans-esophageal echocardiography. At 2 weeks, animals underwent an open chest electrophysiological study; conduction velocity (CV) when pacing at 150ms cycle length (CL), effective refractory periods (ERP) and AF vulnerability were measured. The mean plasma concentration of GAP-134 was 557 ± 239 nmol/L. GAP-134 increased CV (395.1 ± 63.2 vs 307.8 ± 54.6 mm/s, p<0.01), and shortened ERP at 200ms CL (104.0 ± 8.6 vs 112.8 ± 11.5 ms, P<0.05). GAP-134 significantly reduced AF inducibility [% burst attempts inducing AF] and maintenance [mean AF duration, number of episodes >10min] in dogs with less than 100% ΔLASA (n=5). In dogs with more structural remodeling (ΔLASA ≥100%, n=7), CV increased but AF inducibility was unaffected.

Conclusions:
Oral GAP-134 prevents CV slowing in a dog model of atrial cardiomyopathy, but attenuates AF inducibility and maintenance only in dogs with less mechanical remodeling.