bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 524: Influenza Vaccination Reduces The Risk Of Venous Thromboembolism
Authors: Zhu Tienan , Carcaillon Laure , Martinez Isabelle , Cambou Jean-Pierre , Kyndt Xavier , Rivron-Guillot Karine , Vergnes Marie-Christine , Scarabin Pierre-Yves , EMMERICH Joseph
Published: July 2021

It is widely accepted that influenza vaccination reduces the risk of cardiovascular events in patients with coronary artery diseases. However, no information is available concerning the role of influenza vaccination in venous thromboembolism (VTE).

Methods:
A case-control study was conducted in 727 cases and 727 age and sex-matched controls from 11 centers in France (the FARIVE study). Cases were patients with a first documented episode of VTE and without any personal history of cancer within the last 5 years; controls were recruited in the same hospital and had no personal history of venous or arterial thrombosis.

Results:
Overall, 202 (28.2%) of cases and 233 (32.1%) of controls had influenza vaccination during the previous 12 months at the time of recruitment. Information was not available for only 12 subjects out of 1454. The crude odds ratio (OR) for VTE associated with influenza vaccination was 0.76 (95% CI 0.58–0.99). After adjustment for potential confounding variables (age, sex, inclusion date, BMI, educational levels and varicose veins), the OR for VTE associated with vaccination was 0.74 (95% CI 0.57–0.97). The protective effect of vaccination toward VTE was higher in the population below 52 years (median of age), with an OR of 0.52 (95% CI 0.32–0.85). In women below the age of 51, the crude OR for VTE associated with influenza vaccination was 0.50 (95% CI 0.24–1.05) and was significant after adjustment for confounding variables including oral contraceptives, with an OR of 0.41 (95% CI 0.19–0.92). The protective effect of vaccination was similar for different types of VTE (DVT or PE). There was no statistical differences in the protective effects when we compared provoked with unprovoked VTE events.

Conclusion:
This is the first study that demonstrates a protective effect of influenza vaccination in VTE.
In vitro
influenza induces activation of coagulation and inflammation but we cannot rule out that the protective effect of influenza vaccination was due to other unknown mechanisms, as the protective effect was similar within the different seasons of the year. Influenza vaccination may reduce the risk of VTE by 26%. This relationship between influenza vaccination and VTE and its underlying mechanism still need to be analyzed and confirmed in further studies.
Abstract 1359: Only the Catalytic p110alpha Isoform Mediates Class IA PI 3-Kinase-Induced Atherogenic Signals in Vascular Smooth Muscle Cells
Authors: Vantler Marius , Mustafov Lenard , Caglayan Evren , Rosenkranz Stephan
Published: July 2021

Proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMC) are pivotal determinants of the pathogenesis of vascular diseases, which are mainly controlled by growth factor dependent activation of PI 3-Kinase (PI3K). Growth factors like platelet-derived growth factor (PDGF) activate class IA PI3Ks containing one of three p110 catalytic subunits (p110alpha, p110beta, and p110delta). We investigated the specific function of these isoforms for PDGF-controlled proliferation, migration, and apoptosis of VSMC using novel isoform-specific inhibitors. PDGF-dependent proliferation and migration solely depended on p110alpha. Stimulation of VSMC with PDGF-BB (50 ng/ml) mediated a 2.5±0.4 increase (
p
<0.05) of DNA-synthesis (BrdU incorporation assay) and induced a 3.4+/−0.7 fold increase (
p
<0.05) of VSMC migration (modified Boyden-chamber). Inhibition of p110alpha with PIK075 (1
μ
M, Ki=100 nM) completely abrogated PDGF-dependent DNA-synthesis and migration (
p
<0,05), whereas inhibitors against p110beta (TGX 221, 1
μ
M) or p110delta (IC87114 1
μ
M) had no influence. Consistently, PDGF-induced DNA-synthesis and migration were suppressed by siRNA-dependent downregulation of p110alpha (
p
<0,05) whereas p110beta or p110delta knockdown had no effect. Interestingly, stimulation of VSMC with PDGF-BB (50 ng/ml) induced anti- or proapoptotic effects depending on the duration of PDGFR activation. Incubation of VSMC with H
2
O
2
(50
μ
M, 16h) led to a 2.8±0.7 fold increase (
p
>0.05) of apoptosis (Cell Death Detection ELISA). Simultanous addition of PDGF-BB (50 ng/ml) significantly diminished the H
2
O
2
-induced apoptosis (52±7%,
p
>0.05). In contrast, prestimulation with PDGF-BB 24h prior to the addition of H
2
O
2
led to an increase of H
2
O
2
-induced apoptosis (7.8±1.3,
p
>0.05). The anti- as well as the proapoptotic effect depended strictly on p110alpha as PIK075 (1
μ
M,
p
<0,05) or p110alpha specific siRNA completely abrogated PDGF-BB-mediated pro- as well as antiapoptotic effects. Our results demonstrate that only the catalytical PI3K subunit p110alpha mediates the growth factor-induced atherogenic responses. Therefore, p110alpha represents an interesting therapeutic target for prevention of atherosclerosis and restenosis formation.
Abstract 1360: A Potential Role for Nuclear-Delimited Signaling via Nuclear-Envelope Angiotensin Receptors in the Control of Cardiac Gene Expression
Authors: Tadevosyan Artak , Allen Bruce G , Nattel Stanley
Published: July 2021

Angiotensin-II (A-II) regulates cardiac homeostasis via AT1 (AT1R) and AT2 (AT2R) receptors. A-II mitogenic and growth promoting effects contribute to various cardiac remodeling paradigms, largely by altering gene expression. Here, we assessed the possibility that A-II effects on gene expression may be mediated by intracellular A-II acting on AT1Rs or AT2Rs in the nuclear envelope. Western blots of nucleus-enriched rat heart fractions indicated the presence of AT1R and AT2R proteins that preferentially copurified with a nuclear-membrane marker (Nup 62) but not markers of plasma (Calpactin I), Golgi (GM130) or endoplasmic-reticulum (GRP78) membranes. Confocal immunoflorescent microscopy of adult rat and dog cardiomyocytes revealed AT1R and AT2R proteins on nuclear membranes and T tubules. Microinjection of AngII-Alexa488 into single cardiomyocytes indicated preferential binding of A-II to nuclear
vs
cytoplasmic sites (197 ± 7*
vs
110 ± 7 respectively; *p<0.05). Ca
2+
i
-recordings on nuclear preparations (Fura-2 AM) revealed AT1R-mediated Ca
2
+
release (e.g. 17 ± 2 nM control, 264 ± 13 nM* Ang-II and 177±6 nM* for the AT1-selective agonist L-162313). AT1R ligands enhanced
de novo
RNA synthesis in isolated cardiac nuclei incubated with [
α
32
P]-UTP (eg 45±1 CPM/ng DNA control vs 244±3 CPM/ng DNA* with A-II, N=6/group, P<0.001). NF
κ
B mRNA expression (RT-qPCR, relative to
β
-actin) was enhanced by A-II or L-162313 applied to isolated cardiac nuclei, a response that was suppressed by co-administration of valsartan (AT1-selective antagonist). Dose-response experiments with A-II applied to purified nuclei vs whole myocytes showed a 1.5-fold* greater increase in NF
κ
B mRNA levels at saturating concentrations with similar EC50 values, suggesting preferential nuclear signaling. Cardiac nuclear membranes possess AT2Rs and AT1Rs that are coupled to nuclear signaling pathways and regulate transcription. Intracellularly synthesized A-II signaling through cardiac nuclear envelopes may be particularly important for A-II regulation of cardiac gene expression, suggesting potential differential actions of A-II from intra- vs extracellular sources, with important mechanistic and therapeutic implications.
Abstract 1379: Angiotensin II Inhibits the Na+-K+ pump via PKC-dependent Activation of NAD(P)H Oxidase
Authors: Figtree Gemma A , White Caroline N , Liu Chia Chi , Garcia Alvaro , Hamilton Elisha J , Chia Karin K , Rasmussen Helge H
Published: July 2021

Background:
The sarcolemmal Na
+
-K
+
pump, essentially the only export route for Na
+
and pivotal for cardiac myocyte function, is inhibited by angiotensin II (Ang II). Since Ang II activates NAD(P)H oxidase we tested the hypothesis that NAD(P)H oxidase mediates Ang II-induced pump inhibition. This is important for our understanding of pathophysiology and treatment of heart failure, a condition with increases in circulating Ang II levels; intracellular myocyte Na
+
; NAD(PH oxidase activity; and myocardial oxidative stress. The raised myocyte Na
+
is believed to contribute to the electro-mechanical phenotype of contractile abnormalities and cardiac arrhythmias.

Methods and results:
Exposure to 100 nmol/L Ang II increased fluorescence of isolated rabbit ventricular myocytes loaded with a superoxide-sensitive dye. The increase was abolished by pegylated superoxide dismutase (SOD), by the NAD(P)H oxidase inhibitor apocynin and by myristolated inhibitory peptide to
ϵ
-protein kinase C (
ϵ
PKC), previously implicated in Ang II-induced Na
+
-K
+
pump inhibition. Exposure of voltage-clamped myocytes to Ang II decreased electrogenic Na
+
-K
+
pump current, I
p
, from 0.48 ± 0.02 pA/pF, N = 11 to 0.36 ± 0.03 pA/pF, N = 11 (P < 0.05). The decrease was abolished (P < 0.05) by inclusion in pipette solution of: SOD, the gp91ds inhibitory peptide that blocks assembly and activation of NAD(P)H oxidase and by
ϵ
PKC inhibitory peptide. Since co-localization should facilitate NAD(P)H-oxidase dependent regulation of the Na
+
-K
+
pump we examined if there is physical association between the pump and NAD(P)H oxidase. Cell lysate was immunoprecipitated with antibody to the
α
1
pump subunit. Immunoblotting demonstrated co-immunoprecipitation of the
α
1
subunit with caveolin 3 and with membrane-associated p22

phox

and cytosolic p47

phox

NAD(P)H oxidase subunits at baseline. Ang II had no effect on
α
1
/caveolin3 or
α
1
/p22

phox

interaction, but increased
α
1
/p47

phox

co-immunoprecipitation.

Conclusion:
Ang II is known to induce PKC-dependent phosphorylation and translocation of p47

phox

resulting in activation of NAD(P)H oxidase, and we propose this activation occurs in a caveolar microdomain and that it mediates the inhibition of the Na
+
-K
+
pump by Ang II.
Abstract 1383: Mitochondrial Tert Enhances Mitochondria Functions In Vivo by Protecting Mitochondrial DNA Integrity from Oxidative Damage
Authors: Buechner Nicole , Dröse Stefan , Jakob Sascha , Brandt Ulrich , Altschmied Joachim , Haendeler Judith
Published: July 2021

Mitochondria are essential for respiration and oxidative phosphorylation. Mitochondrial dysfunction due to aging processes is involved in pathologies and pathogenesis of a series of cardiovascular disorders. New results accumulate showing that the enzyme telomerase with its catalytic subunit telomerase reverse transcriptase (TERT) has a beneficial effect on heart functions. The benefit of short-term running of mice for heart function is dependent on TERT expression. TERT can translocate into the mitochondria and mitochondrial TERT (mtTERT) is protective against stress induced stimuli and binds to mitochondrial DNA (mtDNA). Because mtDNA is highly susceptible to damage produced by reactive oxygen species (ROS) which are generated in close proximity to the respiratory chain, the aim of this study was to determine the functions of mtTERT in vivo and in vitro. Therefore, mitochondria from hearts of adult, 2nd generation TERT-deficient mice (TERT −/−) and wt littermates were isolated and state 3 respiration was measured. Strikingly mitochondria from TERT −/− revealed a significantly lower state 3 respiration (TERTwt: 987 +/− 72 pmol/s*mg vs. TERT−/−: 774 +/− 38 pmol/s*mg, p<0.05, n=5). These results demonstrated that TERT −/− mice have a so far undiscovered heart phenotype. In contrast mitochondria isolated from liver tissues did not show any differences. To get further insights in the molecular mechanisms, we reduced endogenous TERT levels by shRNA and measured mitochondrial reactive oxygen species (mtROS). mtROS were increased after ablation of TERT (scrambled: 4.98 +/− 1.1 % gated vs. shTERT: 2.03 +/−0.7 % gated, p<0.05, n=4). We next determined mtDNA deletions, which are caused by mtROS. Semiquantitative realtime PCR of mtDNA deletions revealed that mtTERT protects mtDNA from oxidative damage. To analyze whether mitochondrial integrity is required to protect from apoptosis, vectors with mitochondrially targeted TERT (mitoTERT) and wildtype TERT (wtTERT) were transfected and apoptosis was measured. mitoTERT showed the most prominent protective effect on H2O2 induced apoptosis. In conclusion, mtTERT has a protective role in mitochondria by importantly contributing to mtDNA integrity and thereby enhancing respiration capacity of the heart.
Abstract 1419: Biocompatible 3-Dimensional RGD-Modified Alginate Scaffold Designed to Enhance Cell Transplantation to Infarcted Myocardium
Authors: Sondermeijer Hugo P , See Fiona , Seki Tetsunori , Witkowski Piotr , Woodland David C , Hardy Mark A , Itescu Silviu
Published: July 2021

Introduction Cell transplantation strategies for cardiac repair are currently limited by poor survival of transplanted cells. Three dimensional scaffolds improve cell transplant survival after delivery to infarcted myocardium. We aimed to design a biocompatible porous 3D scaffold using custom purified alginate that augments cell viability. Two-dimensional surfaces coated with cyclic RGDfK peptides have been shown to improve cell viability. We hypothesized that covalent attachment of cRGDfK peptide to an alginate scaffold would improve cell viability in 3D. Methods Cyclic RGDfK peptide modified porous scaffolds were fabricated by freeze gelation and subsequently seeded with 3x10e6 neonatal rat cardiomyocytes or 3x10e6 neonatal rat cardiac fibroblasts. Cell containing scaffolds were cultured for one week and cell viability was determined by trypan blue exclusion. Scaffolds were implanted intramuscularly in rats (n=4) for 1 month to determine biocompatibility by multinucleated giant cell formation, compared to immunogenic suture material. Efficacy of circular scaffold (16 mm x 0.75 mm) application to the epicardial surface of rat hearts (n=12) was evaluated macroscopically and by ED-2 positive macrophage staining 1 week after transplantation. Results Scaffolds with interconnected pores (100–200 μm) allowed seeding and even distribution of cells. At 1 week, neonatal rat myocyte viability inside scaffolds increased from 3.3±1.2% (0 mg/g cRGDfK) to 12.3±0.1% (10 mg/g cRGDfk) to 28.9±7.3% (10 mg/g cRGDfk + gelatin) (P<0.05). Clusters of beating myocytes could be detected. Neonatal rat cardiac fibroblast viability increased from 48.8±21% (0 mg/g cRGDfK) to 77.2±3.2% (10 mg/g cRGDfK) (P<0.05). One month after intramuscular scaffold implantation, 1.2±0.7 multinucleated cells could be seen per HPF throughout the scaffold, vs 17.5±4.2 around immunogenic sutures (P<0.05). Scaffolds integrated with the epicardial surface 1 week after transplantation. ED-2 staining was negative. Conclusion Three dimensional scaffolds generated from custom purified alginate modified with cRGDfK peptide promote cardiac cell viability in vitro. Scaffolds are biocompatible and may be used to transplant cells to the myocardium by epicardial application.
Abstract 1427: Pathogenesis of Hypertrophic Cardiomyopathy Caused by the Myozenin 2 Mutations Involves Calcineurin-Dependent and -Independent Mechanisms
Authors: Chen Suet Nee , Lombardi Raffaella , Rodriguez Gabriela , Marian Ali J
Published: July 2021

Identification of myozenin-2 mutations (MYOZ2) has expanded the spectrum of causal mutations for hypertrophic cardiomyopathy (HCM) to include the Z disk proteins. The molecular mechanism(s) by which MYOZ2 mutations cause HCM are unknown. MYOZ2 is known to inhibit calcineurin phosphatase (PP2B). We posit mutant MYOZ2 lose their inhibitory effects on PP2B and hence, the ensuing cardiac phenotype results from enhanced activity of PP2B. To test this hypothesis, we generated lines of transgenic mice expressing either N-terminally flag-tagged wild type (MYOZ2-WT) or mutant MYOZ2-P48 or MYOZ2-M246 regulated by the α-myosin heavy chain promoter. Expression levels of the transgenic and endogenous MYOZ2 proteins were detected by immunoblotting using pan-specific anti-MYOZ2 and anti-Flag antibodies. The transgene proteins constituted 15 to 35% of the total MYOZ2 protein. The total MYOZ2 levels remained unchanged. Immunofluorescence staining showed both WT and mutant MYOZ2 proteins were incorporated into the Z disks. To determine the effects of the WT and mutant MYOZ2 proteins on PP2B activity, we measured PP2B phosphatase and Nuclear Factor of Activated T Cells (NFATc), a downstream target of PP2B, activities in myocardial protein extracts by ELISA-based assays. Expression of MYOZ2-WT reduced PP2B phosphatase and NFAT activities by ~90% and 20%, respectively while equal levels of expression of the mutant MYOZ2 reduced these activities by ~ 50% and 10%, respectively, i.e., a 2-fold reduction in inhibitory effect of the mutant as compared to WT. The mutant MYOZ2-P48 and MYOZ2-M246 showed increased mortality, cardiac hypertrophy, preserved cardiac systolic function, increased myocytes size and interstitial fibrosis. The MYOZ2-WT mice also exhibited cardiac hypertrophy and interstitial fibrosis. Thus, the mutation impart a loss of function effect on MYOZ2, suggesting a possible gain of function of PP2B in humans with with autosomal dominant HCM. However, excess inhibition of PP2B in the heart, as observed in the MYOZ2-WT mice, also led to cardiac hypertrophy and fibrosis. Thus both excess inhibition as well as relative enhancement of PP2B activity in the heart could cause cardiac hypertrophy and fibrosis.
Abstract 1443: Systems-Based Analysis of HDL Metabolism in a Mouse Intercross between Strains CAST and C57BL6/J (CxB)
Authors: Mehrabian Margarete , Farber Charles , Langfelder Peter , Ghazalpour Anatole , Zhou Zhiqiang , Aten Jason , Castellani Lawrence , Drake Thomas , Schadt Eric , Lusis Aldons
Published: July 2021

A recent meta-analysis of three large genome-wide association studies for HDL cholesterol levels revealed several highly significant associations, but altogether these explained less than 10% of the population variance of HDL. Since HDL levels are highly heritable, with heritability estimated at 50–70% in many studies, there are clearly many additional genes, and probably complex genetic and environmental interactions, involved in HDL metabolism. Thus, if “personalized medicine” is to become a reality, these complex factors must be addressed. Combined genetic-genomic approaches have rejuvenated the analysis of complex traits using mouse models, and here report an integrative genomic study of HDL in a large mouse cross. We previously reported the identification of loci associated with HDL cholesterol concentrations using a CXB F2 intercross. We have now generated a much larger CXB cross, consisting of 438 mice, and have integrated genome wide gene expression analysis of liver and adipose with quantitative trait locus (QTL) mapping and causality modeling. These studies were carried out on mice fed a low fat, chow diet and then switched to a high fat, ’Western’ diet. QTL analysis on the clinical traits using R/QTL (http://cran.r-project.org/) revealed a complex inheritance pattern with significant LOD scores at 9 loci, on chromosomes 1,2,4,5,8,9,10,16,18. Of these loci, 6 (chr: 1,4,5,10,16,18) were seen to be involved in genetic-dietary regulation of HDL cholesterol. Expression QTLs (eQTL) were determined using Agilent microarrays for 23,624 transcripts. Genes expressed within a 1-LOD support interval or correlated with HDL (p<2.7E-11) in both adipose and liver were identified. Using Network Edge Orienting (NEO) methods, causal relationships between the identified genes, related QTL peak markers and HDL levels were accessed. The genes were then ranked based on the NEO scores. In liver the highest ranked genes were associated with mitochondrial, ER and golgi trafficking. In adipose, on the other hand, pathways associated with cell signaling, transcription regulation and protein ubiquitation were predicted to be causal for HDL levels. In conclusion, our results reveal a large number of novel pathways and candidate genes for plasma lipid metabolism.

This research has received full or partial funding support from the American Heart Association, AHA Western States Affiliate (California, Nevada & Utah).
Abstract 1451: Hyperpolarization Induces Differentiation Of Human Cardiomyocyte Progenitor Cells
Authors: van Vliet Piet , de Boer Teun P , van der Heyden Marcel A , Sluijter Joost P , Doevendans Pieter A , Goumans Marie-Jose
Published: July 2021

Background: Recently, we have isolated cardiomyocyte progenitor cells (hCMPCs) from human fetal and adult hearts. These cells differentiate into spontaneously beating cardiomyocytes when stimulated with 5-azacytidine. Subsequent stimulation by TGFβ enhances differentiation efficiency to nearly 100%. The underlying molecular mechanisms mediating this cardiomyogenic differentiation are not understood. In skeletal myoblasts, hyperpolarization-mediated activation of calcineurin signaling is crucial for myogenic differentiation. In hCMPCs, whole-cell patch clamp recordings showed a hyperpolarized membrane potential after stimulation with TGFβ or BMP. We hypothesized that hyperpolarization and calcineurin signaling regulate cardiomyogenic differentiation of hCMPCs after TGFβ stimulation.

Methods & Results: To test whether hyperpolarization initiates cardiomyogenic differentiation, hyperpolarization was induced by 1) co-culture of hCMPCs with HEK 293 cells overexpressing a Kir2.1GFP fusion protein (KWGF cells) or 2) culture of hCMPCs overnight in medium containing low potassium concentrations. During co-culture, Lucifer Yellow dye injection in KWGF cells spread to neighboring hCMPCs, indicating cellular coupling. This resulted in stable hyperpolarization in hCMPCs, which could be blocked by addition of the gap junction inhibitor halothane. After two weeks, qPCR analysis revealed increased expression of cardiac sarcomeric genes in the hCMPCs in a dose-dependent manner. Induction of hyperpolarization by culturing hCMPCs with low potassium concentrations also resulted in increased expression of cardiac genes and the formation of spontaneously beating cells. Immunofluorescence staining revealed striated patterns of troponin I and α-actinin. Interestingly, hyperpolarization also increased intracellular calcium levels in hCMPCs, as measured by ratiometric imaging of indo-1 fluorescence, and, subsequently, a time-dependent increase in NFAT-Luciferase reporter activity, indicating activation of the calcineurin pathway.

Conclusion: TGFβ and/or BMP-mediated hyperpolarization of hCMPCs induces calcineurin-mediated cardiomyogenic differentiation.
Abstract 1457: IGFBP-4-Mediated Wnt Inhibition is Required for Cardiac Myogenesis
Authors: Zhu Weidong , Shiojima Ichiro , Zhi Li , Ikeda Hiroyuki , Yoshida Masashi , Naito Atsuhiko T , Minamino Tohru , Nagai Toshio , Komuro Issei
Published: July 2021

Insulin-like growth factor-binding proteins (IGFBPs) bind to and modulate the actions of insulin-like growth factors (IGFs). Although some of the effects of IGFBPs appear to be independent of IGFs, the precise mechanisms of IGF-independent actions of IGFBPs are largely unknown. In this study we demonstrate that IGFBP-4 is a novel cardiogenic growth factor. IGFBP-4 enhanced cardiomyocyte differentiation of P19CL6 embryonal carcinoma cells and embryonic stem (ES) cells in vitro. Conversely, siRNA-mediated knockdown of IGFBP-4 in P19CL6 cells or ES cells attenuated cardiomyocyte differentiation, and morpholino-mediated knockdown of IGFBP-4 in Xenopus embryos resulted in severe cardiac defects and complete absence of the heart in extreme cases. We also demonstrate that the cardiogenic effect of IGFBP-4 was independent of its IGF-binding activity but was mediated by the inhibitory effect on canonical Wnt signaling. IGFBP-4 physically interacted with a Wnt receptor Frizzled 8 (Frz8) and a Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6), and inhibited the binding of Wnt3A to Frz8 and LRP6. Moreover, the cardiogenic defects induced by IGFBP-4 knockdown both in vitro and in vivo was rescued by simultaneous inhibition of canonical Wnt signaling. Thus, IGFBP-4 is an inhibitor of the canonical Wnt signaling, and Wnt inhibition by IGFBP-4 is required for cardiogenesis. The present study provides a molecular link between IGF signaling and Wnt signaling, and suggests that IGFBP-4 may be a novel therapeutic target for heart diseases.
Abstract 1469: Segregation of Copper-dependent Transcription Factor and Copper Chaperone Function of Antioxidant-1 in Wound Healing
Authors: Kim Ha Won , Yang Jay , Finney Lydia , Vogt Stefan , McKinney Ronald D , Wilgus Traci A , DiPietro Luisa A , Ushio-Fukai Masuko , Fukai Tohru
Published: July 2021

Copper plays an essential role in cell growth involved in wound repair and angiogenesis; however, underlying molecular mechanism remains unclear. We recently have found that the copper binding protein antioxidant-1 (Atox1), previously appreciated as a copper chaperone for secretory copper enzymes at the transGolgi network (TGN), mediates copper-induced cell proliferation in cultured fibroblasts. The role of Atox1 in wound healing in vivo is unknown. Using mouse wound healing model, here we show that Atox1 is highly induced at BrdU-positive nuclei in hair follicles and epidermis in wounding tissues where copper is accumulated, as assessed by X-ray fluorescence microscopy and by ICP-mass spectrometry (2.1-fold increase) as compared to non-wounding skin. Confocal microscopy and cell fractionation analysis demonstrate that Atox1 is predominantly found in cytosol in basal state. Copper or PKC activator PMA promotes translocation of Atox1 to nucleus as well as Atox1 threonine phosphorylation in a time-dependent manner. These copper-induced effects on Atox1 are prevented by a specific PKC inhibitor GF109203X. Luciferase reporter gene assays show that copper increases cyclin D1 promoter by 2.8±0.2 fold, which is blocked by the mutation of Atox1 responsive elements in the promoter and by GF109203X. Gel shift and ChIP assays demonstrate that Atox1 directly binds to the Atox1 responsive element including DNA segment in the cyclin D1 promoter. Atox1 knockout (KO) mice show a marked delay in the rate of wound closure (7.1 day) as compared to wild-type (WT) mice (11.2 day), which is associated with decrease in a number of BrdU-positive proliferating cells and cyclin D1 protein expression. All these defective responses in Atox1 KO mice are rescued by lentivirus-mediated re-expression of nuclear targeted Atox1 and Atox1-WT, but not TGN-targeted Atox1. In summary, copper-dependent transcription factor function of Atox1, but not copper chaperone function, plays a major role in wound healing in vivo through PKC-dependent phosphorylation and binding to the cyclin D1 promoter involved in cell proliferation. These findings provide novel insight into Atox1 as a potential therapeutic target for wound repair process or angiogenesis in response to injury.
Abstract 1473: Identification Of A New NRSF-binding Partner, Zfp90, Which Negatively Regulates The Repression Activity Of NRSF, A Critical Transcriptional Regulator Of Cardiac Fetal Gene Program
Authors: Murakami Masao , Kuwahara Koichiro , Harada Masaki , Nakagawa Yasuaki , Usami Satoru , Kinoshita Hideyuki , Yasuno Shinji , Fujiwara Masataka , Ueshima Kenji , Nakao Kazuwa
Published: July 2021

Neuron-restrictive silencer factor, NRSF is a Zn-finger transcription factor that specifically binds to DNA sequence called NRSE to repress the transcription. NRSF is involved in various biological processes, such as neuronal differentiation, carcinogenesis, and cardiac homeostasis. We previously reported that NRSF regulates the transcription of multiple fetal cardiac genes. Cardiac-specific overexpression of a dominant-negative form of NRSF in mice induced reactivation of fetal cardiac gene program, cardiac dysfunction, and sudden arrhythmic death, suggesting NRSF plays important roles in normal cardiac homeostasis. However, the molecular mechanisms or signaling pathways that regulate the activity of NRSF have not been well understood. In an attempt to clarify the regulators of NRSF, we performed yeast two-hybrid screening using NRSF as bait and identified Zfp90 as NRSF-binding protein. NRSF and Zfp90 co-localize in the nuclear, and Zn-finger domain of NRSF specifically interacted with KRAB domain of Zfp90. To monitor the activity of NRSF sensitively, we made NRSF-responsive reporters using NRSE sequence of BNP promoter and established assay system for estimating the NRSF activity. In this assay system, expression of NRSF repressed the reporter activity and co-expression of Zfp90 restored it. Furthermore the expression levels of Zfp90 were elevated in the hearts with cardiomyopathy. These results suggest that Zfp90 functions as a negative regulator of NRSF and contributes to the genetic remodeling during the development of cardiomyopathy..
Abstract 1491: Notch1 Inhibition Interferes with Differentiation of Glomerular Progenitor Cells into Podocytes
Authors: Maestroni Silvia , Urbanek Konrad , Signoretti Sabina , Sohn Regina Lee , Del Monte Federica , Carrillo-Infante Cynthia , Vitale Serena , Liao Ronglih , Anversa Piero , Zerbini Gianpaolo , Leri Annarosa
Published: July 2021

The objective of this work was to identify a population of glomerular progenitor cells (GPCs) capable of generating podocytes which are specialized epithelial cells that stabilize glomerular capillaries and participate in the barrier function of the glomerular filter. The stem cell antigen prominin-1 was detected in GPCs located within the glomerular tuft of the mouse kidney; these cells expressed also the Notch1 receptor. The Notch1 pathway mediates cell fate decision by regulating the expression of transcription factors that maintain stemness or promote differentiation. Upon ligand binding, the Notch1 receptor is cleaved by γ-secretase and the active intracellular domain of Notch1 (N1ICD) translocates to the nucleus where it triggers transcription of Notch1-dependent genes. A consistent co-localization of N1ICD and the transcription factor WT-1 was demonstrated in synaptopodin-positive cells suggesting that Notch1 activation induced the differentiation of GPCs into podocytes. To strengthen this possibility, experiments were conducted in newborn mice because early postnatally there is a dramatic increase in kidney volume and in the number of glomeruli. The function of Notch1 in the generation of podocytes was established by interfering with the Notch1 pathway through the administration of a γ-secretase inhibitor to neonatal mice; γ-secretase inhibition opposes the release and translocation of N1ICD to the nucleus and the initiation of transcription. Over a period of 4–7 days, treated mice showed a 50% reduction in the number of glomeruli. However, the size of glomeruli was preserved. Importantly, in glomeruli of treated mice, the fraction of WT-1-positive cells that co-expressed N1ICD was decreased 50%. Additionally, podocytes showed an immature phenotype lacking synaptopodin in 30% of glomeruli. Quantitative RT-PCR documented that γ-secretase inhibition attenuated the expression of prominin-1, the early developmental kidney transcription factor Pax2 and the podocyte lineage marker WT-1. In conclusion, we have documented that prominin-1-positive GPCs acquire the podocyte phenotype and the Notch1 pathway controls glomerular number during postnatal development by modulating podocyte differentiation.
Abstract 1503: Knockdown of Mog1 Expression Reduces Sodium Currents by Reducing the Trafficking of Cardiac Sodium Channel Na
V
1.5 to the Cell Membrane
Authors: Chakrabarti Susmita , Yong Sandro , Yoo Shin , Wu Ling , Wang Qing Kenneth
Published: July 2021

The cardiac sodium channel (Na
v
1.5) plays a significant role in cardiac physiology and leads to cardiac arrhythmias and sudden death when mutated. Modulation of Na
v
1.5 activity can also arise from changes to accessory subunits or proteins. Our laboratory has recently reported that MOG1, a small protein that is highly conserved from yeast to humans, is a co-factor of Na
v
1.5. Increased MOG1 expression has been shown to increase Na
v
1.5 current density. In adult mouse ventricular myocytes, these two proteins were found to be co-localized at the intercalated discs. Here, we further characterize the regulatory role of MOG1 using the RNA interference technique. Sodium current was recorded in voltage-clamp mode from a holding potential of −100 mV and activated to −20 mV. In 3-day old mouse neonatal ventricular cells transfected with siRNA against mouse MOG1 decreased sodium current densities (pA/pF) compared to control or scramble siRNA treated cells (−10.2±3.3, n=11 vs. −165±16, n=20 or −117.9±11.7, n=11). A similar reduction in sodium current was observed in mammalian expression system consisting of HEK293 cells stably expressing human Na
v
1.5, by transfecting siRNAs against either human or mouse MOG1 (−41.7±8.3, n=7 or, −82.6±9.6, n=7 vs. −130.6±11.5, n=7; −111.5±8.5, n=7, respectively). Immunocytochemistry revealed that the expression of MOG1 and Na
v
1.5 were decreased in both HEK and neonatal cells when compared to scramble siRNAs or control groups. These results show that MOG1 is an essential co-factor for Na
v
1.5 by way of a channel trafficking. Such interactions between MOG1 and Na
v
1.5 suggest that early localization of MOG1 on the membrane of neonatal cardiomyocytes may be necessary for proper localization and the distribution of Na
v
1.5 during cardiac development.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1507: Disruption of KCNH2 Trafficking May Contribute to Severe Clinical Phenotype of Trafficking Deficient Long-QT Type-1 Mutation KCNQ1-T587M
Authors: Biliczki Peter , Girmatsion Zenawit , Brandes Ralf P , Baro Isabelle , Hohnloser Stefan H , Hebert Terence E , Nattel Stanley , Ehrlich Joachim R
Published: July 2021

Background:
The trafficking deficient long QT syndrome (LQTS)-associated KCNQ1 mutation T587M has previously been associated with a severe clinical phenotype that was not explained by biophysical characteristics of the mutation
per se
.

Objective:
We have previously shown significant interactions between KCNQ1 and KCNH2 and speculated that T587M-KCNQ1 might affect this interaction. We therefore compared the effects of KCNQ1 and T587M-KCNQ1 on KCNH2 trafficking and function.

Methods/Results:
While heterologous co-expression of wildtype (WT) KCNQ1 and KCNH2 in CHO cells increased KCNH2 current (H2+Q1, Fig. A

, *P<0.05) in comparison to KCNH2 expressed alone (H2), co-transfection of KCNH2 with T587M-KCNQ1 failed to increase I
KCNH2
. Immunoblots of specific plasma membrane preparations (Fig. B

) and confocal microscopy indicated increased KCNH2 membrane localization in KCNQ1 co-transfected cells, while total KCNH2 expression remained unaffected. These results indicated a chaperone effect as the potential underlying mechanism. Furthermore, effective co-immunoprecipitation of WT channels occurred. T587M-KCNQ1 also co-precipitated with KCNH2, but the LQTS mutant was retained perinuclearly (Fig. C

) and additionally failed to increase membrane KCNH2 expression (Fig. B

), abolishing the WT KCNQ1 chaperone function.

Conclusions:
Trafficking-deficient T587M-KCNQ1 lacks the KCNH2 chaperone function that occurs with WT KCNQ1, limiting KCNH2 membrane localization and potentially explaining the malignant LQTS phenotype. Consideration of KCNQ1-KCNH2 interactions may be important for understanding mutation-specific clinical variability in LQTS presentations.
Abstract 1512: Heteromeric Assembly of HCN4 and C-Terminal Truncated HCN2 Subunits in the Generation of Cardiac Pacemaker (If) Channels
Authors: Ye Bin , Nerbonne Jeanne
Published: July 2021

In sino-atrial and atrio-ventricular nodal cells, hyperpolarization- activated cyclic nucleotide-gated (HCN) cationic channels are expressed that carry inward currents,
I
f
, contributing importantly to the diastolic depolarization critical for cardiac pacemaker functioning. Although previous studies have demonstrated myocardial expression of HCN2 and HCN4 subunits, the roles of these subunits in the generation of mature, functional
I
f
channels remains unclear. In preliminary experiments, completed on transiently transfected HEK293 cells expressing wild type and dominant negative mutant HCN2 and HCN4 subunits, we find that HCN2 and HCN4 preferentially co-assemble to form heteromeric channels. In further studies focused on defining the molecular compositions of functional cardiac
I
f
channels, we developed and validated C-and N-terminal HCN2 and HCN4 subunit specific antibodies and exploited these to identify the HCN subunits expressed in adult (mouse) heart and to immunoprecipitate cardiac HCN-encoded
I
f
channel complexes. These experiments revealed that the HCN2 and HCN4 proteins co-immunoprecipitate from heart, In addition, Western blot experiments revealed that, although the full length HCN2 (105Da) and HCN4 (160Da) proteins are expressed in transiently transfected HEK293 cells and in adult (mouse) brain, the molecular weight of the HCN2 protein in the heart is reduced to about 60 kDa. In addition, the mature, 60 kDa HCN2 protein lacks the C-terminal cAMP binding domain. These observations likely explain the results of previous studies demonstrating that the targeted deletion of HCN2 does not affect the cAMP sensitivity of
I
f
. In addition, taken together, these results demonstrate that functional myocardial
I
f
channels reflect the heteromeric assembly of HCN2 and HCN4 subunits and further that the HCN4 subunit regulates cAMP-mediated regulation of
I
f
channels.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1527: Lipopolysaccharides Up-regulate Kir6.1/SUR2B Channel Expression And Enhance Vascular K
ATP
Channel Activity Through NF-κB And PKA Pathways
Authors: Shi Weiwei , Wu Zhongying , Cui Ningren , Zhang Shuang , Zhu Daling , Jiang Chun
Published: July 2021

Septic shock, characterized by vasodilatation and hyporeactivity to vasoconstrictors, is a major cause of death. Recent studies have shown that an ATP-sensitive K
+
(K
ATP
) channel plays a critical role in septic susceptibility. However, the mechanisms underlying K
ATP
channel responses to sepsis are still unclear. Thus, we performed these studies to demonstrate how the vascular K
ATP
channel changes its activity and expression with an exposure to lipopolysaccharides (LPS), a major septic pathogen. In isolated rat mesenteric arterial rings, phenylephrine (PE) induced concentration-dependent vasoconstriction. At the maximum effect, pinacidil, a selective K
ATP
channel opener, relaxed the rings almost completely. The PE response was almost completely lost after a pretreatment with LPS (1μg/ml) overnight. The LPS treatment produced hyperpolarization of acutely dissociated aortic smooth myocytes. Consistently, LPS treatment augmented K
ATP
channel activity via increasing channel density in plasma membranes in these cells. Quantitative PCR analysis showed that LPS stimulated Kir6.1 and SUR2B expressions in a dose-dependent manner. LPS in 0.01 μg/ml raised Kir6.1 mRNA expression by ~1.8-folds (P<0.01, n=8). Stronger effects were seen with higher concentrations (by 2.5 and 3.0-folds with 0.1 and 1 μg/ml LPS, respectively). SUR2B expression also increased dose-dependently, though to a less degree. The LPS-induced expression of Kir6.1 and SUR2B subunits was suppressed by actinomycin D (2 μg/ml) and actidione (2 μg/ml), suggesting both transcriptional and translational mechanisms are involved. The effect of LPS on Kir6.1 and SUR2B expression was diminished by a pretreatment with the selective NF-κB inhibitors dimethyl fumarate (0.1 mM) or pyrrolidine dithiocarbamate (0.1 mM). Furthermore, the specific PKA blockers Rp-cAMP (100 μM) or KT5720 (1 μM) suppressed LPS-induced Kir6.1 and SUR2B expression. These results therefore suggest that K
ATP
channel activity is enhanced with LPS exposure leading to hyperpolarization of vascular smooth muscles and vasodilation, and such an effect involves up-regulation of Kir6.1 and SUR2B expressions, in which NF-κB and PKA-dependent intracellular signaling pathways may play a role.
Abstract 1531: SCN5A Promoter and Enhancer Variants associated with Atrial Fibrillation
Authors: Watanabe Hiroshi , Darbar Dawood , Atack Thomas C , Yang Ping , Ritchie Marylyn D , Turner Stephen , Ingram Christiana R , Roden Dan M
Published: July 2021

We have recently reported that 5.9% of patients with atrial fibrillation (AF) have rare variants in the coding region of the predominant cardiac sodium channel gene
SCN5A
. In this study, we tested the hypothesis that AF susceptibility is also influenced by variants in the
SCN5A
core promoter and short non-coding nucleotide sequences highly conserved across species (CNS) that we previously identified. We resequenced the
SCN5A
core promoter region (~3 kb) and CNS regions in intron 1 (<1 kb for each) in 376 patients with AF (111 patients with lone AF and 234 patients with AF and cardiac disease) and 47 ethnically-defined controls. The promoter activity of variants showing different frequencies in patients with AF compared to controls was studied using promoter-reporter assays in CHO cells and in neonatal mouse ventricular myocytes.
Identification of variants
Four common variants were identified in the core promoter in controls, and one of the variants (c.−225– 834 T>C) was significantly more common in patients with AF than in controls (minor allele frequency: lone AF, 17%; AF and heart disease, 17%; control, 7%; P<0.05 vs. control for each). In addition, 10 novel rare promoter variants were identified in patients with AF and were absent in controls. In CNS28, a 406 bp region in intron 1 that includes three TEF-1 binding sites, one variant (c.−53±15307 C>T) which is predicted to disrupt one of the TEF-1 binding sites was identified in 7 out of 376 patients with AF (1 with lone AF, 6 with AF and heart disease), but not in controls.
Functional study
There was no difference in promoter activity between wild-type and c.−225– 834 T>C
SCN5A
core promoters expressed in CHO cells. Wild-type CNS28 coexpressed with the
SCN5A
core promoter increased reporter activity by 77 ± 14% (P=0.03) in CHO cells and 261 ± 8% (P=0.001) in cardiomyocytes, compared to
SCN5A
promoter alone. However, c.−53+15307 C>T CNS28 showed smaller effects on promoter activity in both CHO cells (52 ± 2 % of wild-type, P<0.05) and cardiomyocytes (62 ± 8%, P<0.05) compared to wild-type CNS28. Variants in the
SCN5A
promoter and an enhancer region in intron 1 were associated with AF. Variability in
SCN5A
transcription may affect AF susceptibility.
Abstract 1563: Alpha1-adrenoceptor Autoantibody from Sera of Patients with Primary Hypertension Induced Vascular Endothelial Cell Injury
Authors: Yan Li , Xu Yanwu , Wu Xiangdong , Yang Guangzhao , Liu Jingyi , Wang Xiaoliang , Ma Xinliang , Liu Huirong
Published: July 2021

Background:
Vascular endothelial dysfunction is recognized as a characteristic of patients with primary hypertension, and accumulating evidence demonstrated that immunologic factor play an important role in the pathophysiology of hypertension. Previous study has shown that high level of alpha1-adrenoceptor autoantibody (alpha1-AA) existed in the sera of patients with primary hypertension and induced significant vasoconstrictive effects on arteries from important organs. However, whether alpha1-AA involves in vascular endothelial cell injury in hypertension remains unknown. Therefore, the aim of our present study is to explore the role of alpha1-AA in vascular endothelial cell injury
in vitro
.

Methods:
Alpha1-AA was purified from sera of patients with primary hypertension, and its purification, concentration and titer were detected by SDS-PAGE, BCA assay and ELISA, respectively. LDH release, Caspase-3, 8, 9 activity, apoptosis index (aridine orange and propidium iodide staining, AO/PI staining) and DNA damage (single cell gel electrophoresis assay, SCGE) in cultured HUVEC were examined after administration of alpha1-AA (1 μM) for 24h, respectively.

Results:
In alpha1-AA administration group, apoptosis index was significantly higher than that in control group(12.83 ± 1.19 %
vs.
2.92 ± 0.57 %,
P
<0.01), caspase-3(2.67±0.21 nmol/h/mg protein
vs.
0.95±0.28 nmol/h/mg protein,
P
<0.01) and caspase-8 (0.58±0.12 nmol/h/mg protein
vs.
0.14±0.06 nmol/h/mg protein,
P
<0.01) activity were increased consistently, however, no remarkable change in caspase-9 activity; meanwhile, the autoantibody also resulted in more than 2.5-fold increase in LDH release (
P
<0.01) and higher tail moment value of SCGE (15.42 ± 1.28 in alpha1-AA group
vs.
1.59 ± 0.29 in control group,
P
<0.01) respectively.

Conclusion:
Our results indicated that alpha1-AA can induce DNA damage, pro-apoptotic and cytotoxic effect on vascular endothelial cell
in vitro
, which may contribute to the vascular injury in patients with primary hypertension. The results suggest that production of alpha1-AA is a novel risk factor in hypertensive patients and this auto-antibody may play an important role in the development of hypertension.
Abstract 1590: Autophagic Processing Of Ubiquitinated Protein Aggregates During Unloading-induced Cardiac Atrophy
Authors: Blagg Andrew G , Zhu Hongxin , Rothermel Beverly A , Hill Joseph A
Published: July 2021

Background:
Mechanical unloading of the diseased ventricle has emerged as an important treatment strategy in advanced heart failure. In rare instances, dramatic improvements in ventricular performance are seen, yet little is known regarding underlying mechanisms. Autophagy is an ancient mechanism of bulk protein degradation recently shown to be activated by a variety of forms of cardiac stress.

Methods and Results:
To test for a role of autophagy in atrophic remodeling of the unloaded left ventricle (LV), murine wild-type (WT) and “autophagy reporter” (αMHC-GFP-LC3 transgenic) hearts underwent heterotopic transplantation into the abdomen of same-strain recipient mice. The donor hearts were thereby mechanically unloaded while the native recipient hearts served as controls. In WT hearts, cardiomyocyte cell surface area declined 27±2% at 7 days. Processing of LC3 (microtubule-associated light chain 3) increased in atrophying donor hearts, indicative of increased autophagosome formation. Further, significant increases in the number of GFP-LC3 puncta in “autophagy reporter” hearts were observed. Levels of the pro-autophagic protein Beclin 1 increased in donor hearts, further evidence of increased autophagic flux during atrophic remodeling. In atrophic donor hearts, we detected significant increases in polyubiquitinated proteins as well as in the abundance of the ER chaperone proteins GRP78/BIP and GRP94, indicative of an unfolded protein response. As autophagy degrades numerous cellular components including polyubiquitinated protein aggregates, we transplanted hearts haploin-sufficient for
beclin 1
, a gene required for early processing of autophagosomes (≈50% decrease in autophagy). In these studies, we detected robust increases in the abundance of polyubiquitin proteins in
beclin 1
±
donor hearts relative to WT (62% vs 12%, p<0.05).

Conclusions:
Autophagy is activated during cardiac atrophy and participates in processing of protein aggregates. Together, these findings implicate autophagic activity in the remodeling of the unloaded LV.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).