bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P322: Chamber-Specific Differences in Oxidative Stress in Newborn Versus Adult Rabbit Hearts Following Ischemia-Reperfusion
Authors: Cabigas E Bernadette , Ding Guoliang , Chen Tao , Saafir Talib , Wagner Mary B , Davis Michael E
Published: July 2021

Each year, tens of thousands of children undergo cardiopulmonary bypass (CPB) to correct congenital heart defects. While necessary for surgery, CPB involves stopping the heart and exposing it to ischemic conditions. While much is known about adult injury, little is known about the effects of global ischemia on newborn ventricles. The purpose of this study was to determine age- and chamber-specific differences in oxidative stress following global ischemia-reperfusion injury. We studied newborn (2–4 days) and adult (>8 weeks) rabbit hearts subjected to Langendorff ischemia-reperfusion (30 minutes ischemia, 60 minutes reperfusion). Catalase and superoxide dismutase (SOD) activities were measured, as well as real-time hydrogen peroxide (H
2
O
2
) and superoxide (O
2
−
) generation using confocal microscopy in isolated left and right ventricular (LV and RV) myocytes exposed to hypoxia. Our data demonstrate chamber and age-specific changes in oxidative stress. During ischemia, H
2
O
2
increased significantly in the RV while remaining constant in the LV of newborn rat rabbit myocytes. In contrast, there was a smaller, nonsignificant increase in H
2
O
2
in both both RV and LV myocytes of adults. The increase seen in the RV of newborns was several fold higher than that of adults (4.3-fold vs. 2.2 fold; p<0.05). In whole heart tissue, catalase activity was significantly increased following ischemia in both adult ventricles, while no increase was seen in newborn hearts compared to sham hearts. Additionally, levels in newborns were several fold lower (p<0.05), indicating less scavenging potential. SOD activity was increased from sham vs. ischemia in the LV of both adult and newborn hearts, but only in the RV of the newborn heart (0.80±0.09 to 3.0±0.43 U/mg; p<0.001). Intra-cardiac injection of an H
2
O
2
scavenger, Ebselen, loaded in nanoparticles, improved recovery of developed pressure (49.53±16.25 to 80.0±11.31) in the RV of the newborn (p<0.05), suggesting the local delivery of an exogenous antioxidant is cardioprotective in newborn RVs. Our data demonstrate there are ventricle-specific differences in oxidative stress between newborn and adult rabbit hearts. Local therapy is able to address some of these differences and may limit damage during bypass surgery.
Abstract P323: Waon Therapy, a Form of Thermal Therapy, Reduces Oxidative Stress Systemically and Inhibits the Progression of Cardiac Dysfunction in TO-2 Cardiomyopathic Hamsters with Heart Failure
Authors: Ikeda Yoshiyuki , Miyata Masaaki , Akasaki Yuichi , Miyauchi Takahiro , Furusho Yuko , Ide Kanako , Hamasaki Shuich , Tei Chuwa
Published: July 2021

Background:
Oxidative stress is one of the most crucial factors that develop chronic heart failure (CHF), leading to cardiac apoptosis and fibrosis and vascular endothelial dysfunction. We have reported that Waon therapy, which is a form of thermal therapy using a far infrared-ray dry sauna at 60 degrees centigrade, improves cardiac and vascular endothelial functions and prognosis in patients with CHF. The aim of this study is to investigate whether Waon therapy reduces oxidative stress and prevents from developing cardiac dysfunction in CHF.

Methods:
Thirty-week old male TO-2 cardiomyopathic hamsters with CHF were divided into Waon therapy or control group. Waon therapy group underwent Waon therapy daily for 4 weeks. Control hamsters did not take any treatment. We examined the amounts of reactive oxygen species of serum, hearts and aortas using ELISA and immunohistochemistry. We measured left ventricular % fractional shortening (LV%FS), and performed TUNEL and Azan staining of hearts to assess cardiac function, apoptosis and fibrosis, respectively. Anti-oxidants and apoptotic and angiogenetic factors were assessed by Western blot. All examinations were performed after 4 weeks of treatment.

Results:
Four-week Waon therapy significantly decreased oxidative stress of serum, hearts and aortas compared to those of controls. Waon therapy significantly increased LV%FS and decreased cardiac apoptosis and fibrosis (LV%FS, Waon therapy: 23.3±4.3 vs. control: 16.5±4.2%, P<0.01, TUNEL positive nuclei, 22.0±2.6 vs. 49.3±7.2%, P<0.01, % fibrosis, 20.6±5.3 vs. 47.6±4.8%, P<0.01). Waon therapy significantly increased the expressions of manganese superoxide dismutase, heat shock protein 27 (HSP27) and HSP32 of hearts and aortas, which negatively modulate oxidative stress, compared to those of controls. Waon therapy significantly increased endothelial nitric oxide synthase and decreased plasminogen activator inhibitor-1 of aortas. In addition, Waon therapy significantly decreased Bax, cleaved caspase 3 and cytochrome c and increased Bcl-2 and hypoxia-inducible factor-1α of the failing hearts.

Conclusions:
Waon therapy reduces oxidative stress systemically and inhibits the progression of cardiac dysfuntion in TO-2 cardiomyopathic hamsters.
Abstract P332: Effects of Intravenous Infusion of Polyunsaturated Fatty Acids and Dextrose on Blood Pressure and Endothelial Function in Obese Subjects
Authors: Gosmanov Aidar R , Smiley Dawn , Siquiera Joselita , Robalino Gonzalo , Peng Limin , Umpierrez Guillermo E
Published: July 2021

Hyperglycemia and elevated free fatty acids (FFAs) are implicated in the development of hypertension and endothelial dysfunction. We recently reported that 8-hour infusion of soy-bean oil containing polyunsaturated fatty acids (Intralipid) results in the elevation of blood pressure (BP) and endothelial dysfunction in obese healthy subjects. However, the effects of dextrose infusion or combination of dextrose and Intralipid on BP, endothelial function and insulin action are not known. Accordingly, we compared the effects of 8-hour infusion of normal saline at 40 ml/hr, Intralipid 20% at 40 mL/hr, dextrose 10% at 40 ml/hr and combination of Intralipid and dextrose on BP, endothelial function in 12 obese healthy subjects [ages:41±7 yrs, BMI:32±2 kg/m
2
, BP:113/65 mmHg, HOMA-IR: 2.0±1.0]. Blood pressure, brachial artery flow-mediated dilatation (FMD), and levels of FFAs, glucose, and insulin were measured at 0, 4 and 8 h of infusion. Intralipid infusion significantly increased BP, decreased FMD, and increased plasma FFAs (Table). Unlike Intralipid alone, the combination of Intralipid and dextrose did not increase BP but resulted in FMD changes similar to Intralipid alone. Levels of plasma glucose and insulin increased over time after dextrose infusion alone or in combination with Intralipid but not with lipid infusion alone. Compared with Intralipid, the addition of dextrose to Intralipid led to restoration of FFAs to normal level.

In summary, Intralipid but not dextrose infusion alone or in combination with Intralipid results in significant elevation in blood pressure in obese healthy subjects. In contrast, dextrose administration had no effect on Intralipid-induced endothelial dysfunction. The mechanisms underlying differences in vascular response after addition of dextrose to Intralipid are not known, but these results indicate that dextrose-induced mild hyperinsulinemia may regulate adverse hemodynamic effects of fat administration in obese subjects.
Abstract 1: Nucleolar Proteins Controlling Cardiac Phenotype in Rat Myocytes and Zebrafish Revealed by Chromatin Proteomics
Authors: Monte Emma , Mouillesseaux Kevin , Chen Haodong , Ren Shuxun , Wang Yibin , Chen Jau-Nian , Vondriska Thomas M , Franklin Sarah
Published: July 2021

Cardiac hypertrophy is a common precursor to heart failure, during which cardiomyocytes grow to compensate for an increased workload. Hypertrophic cardiomyocytes undergo significant changes in cellular plasticity by adopting the expression profile and some phenotypic aspects of more primitive (embryonic or fetal) cardiac cells. These global changes in gene expression, conserved between humans and animal models of heart failure, must be preceded by structural alterations to the chromatin. Specifically, chromatin regions must be architecturally modified to allow or deny access for transcriptional machinery. However, the proteins responsible for remodeling chromatin to accomplish these gene expression changes during cardiac hypertrophy and failure are largely unknown. We used a proteomics approach to identify proteins bound to cardiac chromatin and to quantify changes in their abundance during disease. Quantitative mass spectrometry and bioinformatics revealed that 366 of the chromatin-bound proteins detected in this study displayed altered expression in a mouse model of pressure overload cardiac hypertrophy and failure. This included the chromatin remodeling protein Nucleolin (Ncl), which exhibited increased association with chromatin in the hypertrophic heart. To examine its role in regulating cardiac morphology and function we performed morpholino based knockdown of Ncl in zebrafish embryos. Ncl knockdown promoted the expression of bmp4 (a fetal marker), inhibited normal cardiomyocyte differentiation and resulted in abnormal heart chamber formation and looping. Hearts in surviving fish exhibited functional deficits as measured by fluorescence imaging and line-scanning analysis. To investigate the actions of Ncl in the mammalian cardiomyocyte, knockdown was carried out in isolated rat ventricular myocytes using siRNA. Loss of Ncl induced heterochromatin formation (increased Histone H3 K9-trimethylation), suppressed rDNA transcription (52% decrease in pre-rRNA via qPCR) and promoted fetal gene expression (65% increase in ANF; 41% increase in β-MHC transcripts). Overall, this study identifies Ncl as a regulator of chromatin structure, cell growth via ribosome biogenesis and cellular plasticity in the cardiomyocyte.
Abstract 100: An Altered Epigenomic Mark Predisposes to Dilated Cardiomyopathy Through Alternative Splicing of Sarcomeric Gene Expression in Mice
Authors: Vujic Ana , Robinson Emma , Ito Mitsutero , Haider Syed , Skepper Jeremy , Methner Carmen , Ferguson-Smith Anne , Foo Roger
Published: July 2021

Distinct epigenomic patterns of DNA methylation exist in important elements of the cardiac genome in human end-stage cardiomyopathy but whether these contribute to disease progression is unknown. We examined and found that the main DNA methyltransferase expressed in the adult mammalian heart is DNMT3B. We therefore generated a conditional inducible Dnmt3b knockout mouse to test the effect of altered DNA methylation in the heart. Upon induction in the adult mouse, Dnmt3b knockout (CKO) led rapidly to severe systolic insufficiency and myocardial thinning. Examination of myocyte nuclei reflected a loss of homogeniety and re-distribution of heterochromatin. This was accompanied by widespread myocardial interstitial fibrosis and myofibrillary disarray. We examined the myocardial transcriptome of CKO mice by RNA-seq and discovered significant changes in levels of transcript splicing isoforms especially those of sarcomeric genes. When subjected to pressure overload, CKO mice failed to mount a compensatory hypertrophic response and instead developed accelerated fulminant dilated cardiomyopathy. Our results demonstrate that DNA methylation has a distinct role with regulating transcript isoform splicing apart from gene expression. In particular, we have highlighted a mechanism by which the epigenome may mediate the development and progression of a complex disease.
Abstract 102: Cardiac Sodium/Glucose Cotransporter 1, a Novel Cardiac Glucose Transporter, Is Cardioprotective in Ischemia-Reperfusion Injury
Authors: Ramratnam Mohun , Banerjee Sanjay , Witham William , Sharma Ravi , Ramani Ravi , McGaffin Kenneth R , Ahmad Ferhaan
Published: July 2021

Background:
The expression of cardiac sodium/glucose cotransporter 1 (SGLT1) is up-regulated in ischemic heart disease. However, the regulation and the biological role of SGLT1 activity in cardiomyocytes are unclear.

Hypothesis:
We hypothesize that SGLT1 inhibition during ischemia-reperfusion (IR) will lead to worsening cardiac function and recovery. In addition we hypothesize that SGLT1 expression is upregulated by increased AMPK activity in ischemia.

Methods:
Transgenic mice with cardiac-specific RNAi knockdown of SGLT1 (TG
SGLT1-KD
) were subjected to
ex vivo
IR (n > 3/group) using a Langendorff hanging heart preparation with 15 min of no-flow ischemia and 20 min of reperfusion while undergoing hemodynamic monitoring. FVB strain wild-type 8-12 week-old male mice (n > 3/group) were administered either the SGLT1 inhibitor phlorizin (400 mg/kg IP) or vehicle and subjected to
in vivo
ischemia for 30 min by ligation of the left anterior descending (LAD) coronary artery. Hearts were harvested 48 hours later.
In vitro
studies were conducted in HL-1 cardiomyocytes subjected to 1 h of simulated ischemia with 1 mM NaCN. HL-1 cells were also treated with 1 mM AICAR, an AMPK activator, for 16 hours and SGLT1 expression was measured.

Results:
In
ex vivo
IR studies, there was an impairment in the early recovery of double product in the TG
SGLT1-KD
mice compared to control mice (37% vs 92% at 1 min into reperfusion,
P
< 0.05). In
in vivo
IR studies, phlorizin led to a marked increase in oxidative stress documented by cardiac malondialdehyde (MDA) (control 1.00±0.00; IR 1.55±0.35; IR + phlorizin 16.15±0.40,
P
<0.001),an increase in apoptosis documented by TUNEL staining, and hemodynamic impairment: +dP/dt (mmHg/sec), control 11445±504, IR 9811±2613, IR + phlorizin 5139±481,
P
<0.05; -dP/dt (mmHg/sec), control -10625±589, IR -6770±1142, IR + phlorizin -4153±474,
P
<0.05. In HL-1 cardiomyocytes, there was a 6.7-fold increase in SGLT1 expression after simulated ischemia, and a 2.0-fold increase after AICAR exposure.

Conclusion:
SGLT1 expression increases when cardiomyocytes are subjected to ischemia, an effect that may be mediated by AMPK. Inhibition of SGLT1 leads to impaired myocardial recovery from ischemia.
Abstract 107: The Innate Immune NALP3 Receptor Mediates Cardiac Ischemic Preconditioning in the Isolated Heart
Authors: Zuurbier Coert J , Jong Willeke , Eerbeek Otto , Koeman Anneke , Pulskens Wilco P , Leemans Jaklien C , Hollmann Markus W
Published: July 2021

Objective:
Recent studies indicate that the innate immune system is not only activated by exogenous pathogens and pollutants, but also by endogenous danger signals released during ischemia and necrosis. As activation triggers (cellular K+ extrusion, ROS production) for the innate immune NALP3 inflammasome protein complex overlap with those for cardiac ischemia-reperfusion (I/R) and ischemic preconditioning (IPC), we explored the possibility that the NALP3 inflammasome is involved in IPC and acute I/R injury of the heart.

Methods & Results:
Baseline performance and I/R injury was investigated in isolated, Langendorff-perfused hearts from wild-type, ASC-/- and NALP3-/- mice. Baseline cardiac performance was unaffected by genetic deletion of NALP3 inflammasome components. Deletion of the ASC and NALP3 component exacerbated IR injury on mechanical parameters of the heart. However, deficiency in ASC or NALP3 was without effect on cell death (LDH release during reperfusion). In a separate series we subjected these same hearts to IPC, consisting of 3 x 5 min I/R preceding the 35 min I. IPC protected WT and ASC-/- hearts against IR injury (improved function and less cell death). However, no IPC effects on function or cell death were observed in NALP3-/- hearts. NALP3-/- hearts had significantly decreased cardiac IL-6 levels with a trend towards lower Il-1β levels at end reperfusion, suggesting abrogating of IPC through diminished IL-6 and/or Il-1β signalling. However, subsequent experiments showed that inhibition of the IL-1r receptor with the IL-1 receptor inhibitor Anakinra (100 mg/L) was without effect on IPC in WT hearts. In contrast, neutralising IL-6 using an antibody against IL-6 in WT hearts abrogated IPC in NALP3-/- hearts.

Conclusions:
The data suggest that the innate immune NALP3 protein, in an NALP3-inflammasome-independent fashion, is an integral component of IPC in the isolated heart, possibly through an IL-6 dependent mechanism
Abstract 108: Glycolysis, Cardioprotection and Their Modulation by Differential Interactions of Hexokinase I and II with Mitochondria
Authors: Calmettes Guillaume , John Scott , Weiss James N , Ribalet Bernard
Published: July 2021

Rationale:
Neonatal heart is more glycolytically active and less susceptible to anoxia/reoxygenation injury than adult heart. Hexokinase (HK) is the first step in glycolysis and its binding to mitochondria is known to be cardioprotective. Since neonatal cardiac myocytes mainly express the HKI isoform, whereas HKII predominates in adults, we explored the functional consequences of such difference.

Objective:
To investigate whether differential interactions of HKI and HKII with mitochondria in neonatal (NRVM) and adult rat ventricular myocytes (ARVM) account for their different metabolic profiles and susceptibility to anoxia/reoxygenation injury.

Methods and Results:
We performed real-time imaging in isolated NRVM and ARVM expressing the genetically-encoded biosensors FLIPglu-600µM to track intracellular glucose metabolism and YFP-tagged HK constructs to track subcellular HK localization, together with a propidium iodide-based cell death assay. We show that HKI remains bound to mitochondria in both NRVM and ARVM, while HKII distributes between the mitochondria and cytoplasm. Extracellular glucose removal displaced HKII from mitochondria in ARVM, but not in NRVM, whereas iodoacetate (IAA) displaces HKII in both. These differences were associated with a 35-fold greater glycolytic activity and markedly enhanced resistance to anoxia/reoxygenation injury in NVRM. NRVM became comparably susceptible to anoxia/reoxygenation injury only when treated with IAA to suppress HK interaction with mitochondria (whereas glucose removal alone had no effect).

Conclusions:
HKs exhibit stronger interaction with mitochondria in NRVM, accounting for their enhanced glycolytic activity and increased resistance to anoxia/reoxygenation injury. HKI may be superior to HKII in protecting the heart because of its stronger interaction with mitochondria.
Abstract 11: Transgenic Overexpression of Snf1-Related Kinase in the Heart Improves Cardiac Metabolic Efficiency and Response to Myocardial Ischemia
Authors: Rines Amy , Burke Michael , Wu Rongxue , Abel E Dale , Ardehali Hossein
Published: July 2021

Introduction:
Snf1-related Kinase (SNRK) is a serine/threonine kinase with sequence similarity to AMP-activated kinase. The function and substrates of SNRK are unknown. We have found that SNRK is upregulated in hearts from patients with ischemic cardiomyopathy, and our previous gene array data suggested that SNRK alters metabolic gene expression. Here, we assessed the hypothesis that SNRK regulates cardiac metabolism.

Results:
SNRK transgenic mice with specific overexpression of SNRK in the heart displayed decreased glycolysis (1267.66 vs. 1925.60 nmol/min/g dhw, p=0.004, n=6), glucose oxidation (441.73 vs. 602.68 nmol/min/g dhw, p=0.068, n=6), and palmitate oxidation (262.39 vs. 385.07 nmol/min/g dhw, p=0.005, n=6) compared to wild type littermate controls in perfused working hearts. However, cardiac power output and function were maintained, demonstrating that SNRK transgenic mice have increased metabolic efficiency. Mitochondria isolated from SNRK transgenic mice had increased state 3 respiration and oxidative capacity, along with an increased respiratory control ratio (state 3/state 4 respiration), indicating reduced mitochondrial uncoupling. To identify the mechanism for the effects of SNRK on metabolic flux, we performed a yeast two hybrid screen with SNRK bait. Tribbles homolog 3 (Trb3), an inhibitor of Akt, was identified as a SNRK-interacting protein, which was verified by co-immunoprecipitation and mammalian two hybrid assay. Trb3 protein levels were increased in the SNRK transgenic mice, and phosphorylation of Akt and its substrate GSK3 was reduced. SNRK transgenic mice also had decreased mRNA levels of glucose transporter 1 and 4, hexokinase II, and phosphofructokinase. Additionally, mRNA levels of the fatty acid metabolism regulators peroxisome proliferator-activated receptor (PPAR) α and PPARγ were reduced, as well as their targets uncoupling protein 2 and 3. Finally, SNRK transgenic mice had decreased infarct size compared to wild type littermates (14.35% vs. 20.63% total left ventricle area, p=0.023, n=6) following myocardial infarction for seven days.

Conclusions:
Our results demonstrate that SNRK increases cardiac metabolic and mitochondrial efficiency and resistance to myocardial infarct cell death.
Abstract 117: TWEAK (Tumor Necrosis Factor-Like Weak Inducer of Apoptosis) Impedes Healing After Myocardial Infarction in Mice
Authors: Pachel Christina , Wagner Helga , Bayer Barbara , Dienesch Charlotte , Umbenhauer Sandra , Mathes Denise , Heitzmann Wolfram , Salzmann Steffen , Ertl Georg , Wajant Harald , Frantz Stefan
Published: July 2021

Introduction:
TNF-related weak inducer of apoptosis (TWEAK) triggers multiple cellular responses involved in left ventricular remodelling after myocardial infarction (MI), including cytokine production, inflammation, and wound healing. We therefore hypothesized that TWEAK-Fn14 interactions improve wound healing after experimental MI.

Methods/Results:
We investigated the effects of exogenous TWEAK by applying (1) cross-linked TWEAK - activating the classical NF-kB-signalling pathway, (2) TWEAK and (3) human serum albumin-tagged (HSA)-TWEAK - both activating the alternative NFkB-signalling pathway, or (4) placebo after the induction of experimental MI. Treatment with TWEAK and HSA-TWEAK surprisingly resulted in significantly higher mortality after MI (survival, placebo vs. TWEAK vs. HSA-TWEAK, 66.7 (10 of 15) vs. 14.3 (4 of 26) vs. 7.1 % (1 of 19)) due to cardiac ruptures in the TWEAK- and HSA-TWEAK groups. Ruptures were not related to changes in cardiac architecture since left ventricular dimensions as measured by echocardiography were identical between HSA-TWEAK and the placebo group three days after MI, a time point where cardiac ruptures had not taken place yet. We rule out direct remodelling defects, since MMP9 mRNA expression and proteolytic activity (gelatine zymography), and collagen1α1 mRNA expression were not altered between the groups. HSA-TWEAK induced an exaggerated inflammatory response: Infiltration of neutrophils into the border zone was significantly increased as assessed by immunohistochemistry (placebo vs. HSA-TWEAK, 297.91 vs. 455.75 neutrophils/ mm2, p < 0.001). FACS-Analysis showed a significant up-regulation of CD45+-cells in the infarcted area (CD45+-cells, placebo vs. HSA-TWEAK, 4.52 vs. 9.38 %, p < 0.05). This was associated with the up-regulation of genes important for the recruitment (MIP2, MCP-1 and MCP-5) and differentiation (IL5, IL12 and IFN-γ) of leukocytes (pg/ml, placebo vs. HSA-TWEAK: p < 0.05: MIP2, 1.41 vs. 7.83; IL5, 4.13 vs. 18.75; p < 0.001: MCP-1, N.D. vs. 28.72; MCP-5, N.D. vs. 7.36; IL12, 2.07 vs. 44.51; IFN-γ, N.D. vs. 7.47).

Conclusion:
Treatment with TWEAK reduces survival after experimental MI due to left ventricular rupture. This may be mediated by an exaggerated inflammatory response.
Abstract 119: Targeting Cardiac Hypertrophy with Small Molecule Inhibitors of JMJD2A
Authors: Zhang Qing-jun , Rai Ganesha , Jadhav Ajit , Simeonov Anton , Maloney David , Liu Zhi-Ping
Published: July 2021

One of the major challenges in managing and treating heart failure patients is to develop disease-modifying drugs that can prevent, reverse, or slow down the disease progression. Upon pathological insults, the heart undergoes remodeling processes, including left ventricular hypertrophy and reprogramming of gene expression. Understanding the mechanisms involved could provide a key to develop interventional therapeutics. Epigenetic modification of chromatin, including histone methylation, regulates gene transcription in response to environmental signals. JMJD2A is a trimethyl-lysine specific histone lysine demethylase. To study the role of JMJD2A, we generated heart specific JMJD2A overexpression and deletion mouse lines. Our studies with these genetically modified mice indicated that JMJD2A is required for pathological cardiac hypertrophy. Furthermore, we show that the demethylase activity of JMJD2A is required for its transcriptional activity. These data suggests that targeting JMJD2A enzymatic activity may be used to suppress hypertrophic remodeling. To test this hypothesis, we tested a collection of small molecule inhibitors of JMJD2 in collaboration with Chemists in NIH and identified several small molecule inhibitors of JMJD2A that are active in cell-based assays. These small molecule inhibitors of JMJD2A inhibited the phenylephrine-stimulated cardiomyocyte hypertrophy in vitro. Our data suggests that JMJD2A enzymatic activity may act as a hypertrophic determinant and may be an innovative drug target for prevention and treatment of pathological cardiac hypertrophy and heart failure.
Abstract 12: GRK2 Regulates Enos Level in Endothelial Cells Through a Proteasome-Dependent Mechanism
Authors: Ciccarelli Michele , Franco Antonietta , Anastasio Antonio , Dorn Gerald W , Trimarco Bruno , Iaccarino Guido
Published: July 2021

eNOS is fundamental to regulate vascular functions such vasodilation, permeability and it has been demonstrated that overexpression of the Kinase of G protein coupled receptor 2 (GRK2) in endothelial cell (EC) reduces eNOS activity through AKT. However potential implication of GRK2 inhibition/removal from EC has not been explored yet. For this aim, we used vascular cell isolated from aorta of transgenic mice with GRK2 gene flanked by p-lox sequence (GRK2
flox/flox
). CRE recombinase, introduced in these cells by means of adenovirus infection (AD-CRE), cut and legates at p-lox sequences producing GRK2 gene removal. We observed that GRK2 deletion by AD-CRE produces reduction of eNOS levels as compared to AD-GFP used as control (AD-CRE:0.5±0.01 vs AD-GFP:3.2±0.2, p<0.05). To evaluate if GRK2 deletion produces reduced transcription of eNOS gene, we performed RT-PCR which did not show significant differences between AD-CRE and AD-GFP treated cells in eNOS mRNA level. We then incubated cells with a proteasome inhibitor, MG-132 (10
-5
M, 30’), to explore an involvement of Ubiquitin dependent degradation, and observed a recover of the eNOS level (AD-CRE:3.5±0.11 vs AD-GFP:3.4±0.3, ns). To evaluate if GRK2 regulates eNOS ubiquitination, we pulled down equal amount of eNOS from bovine aortic endothelial cells (BAEC) transfected with a plasmid codifying for GRK2 or a control plasmid, and evaluated levels of ubiquitinated eNOS by western blot. We observed that GRK2 overexpression reduces level of eNOS ubiquitination as compared to control, which is also accompanied by increased level of eNOS. Morever, we transfected BAEC with plasmids expressing GRK2 wild type (GRK2 wt) and GRK2 kinase defective mutant (GRK2K220R). We found that both GRK2 wt and K220R are able to induce eNOS accumulation in BAEC (GRK2: 4.5±0.3, GRK2K220R: 4.8±0.4 vs Control: 2.1±0.1, p<0.05) indicating a kinase independent mechanism of eNOS ubiquitination and degradation. More experiments are needed to evaluate mechanisms through which GRK2 protects eNOS from Ubiquitin mediated degradation, nevertheless, these data indicate a novel mechanism of regulation of eNOS levels that can have important implications in the overall cellular physiology and pathology.
Abstract 120: Notch Signaling Negatively Regulates Vascular Endothelial Cell Senescence
Authors: Yoshida Yohko , Minamino Tohru
Published: July 2021

Accumulation of senescent vascular cells occurs in aged vessels, which leads to an increase of inflammation and a decline of regenerative potential, thereby promoting vascular dysfunction and atherosclerosis. However, the molecular mechanisms of these age-related changes remain unclear. The Notch pathway is a highly conserved signaling that controls cell fate determination and differentiation during the development of various tissues. In adults, Notch signaling has been reported to be essential for neovascularization and is implicated with the age-associated conditions such as cancer, neuronal disorder and impaired regeneration of skeletal muscle. Here, we show that Notch signaling has a crucial role in endothelial cell senescence. We found that inhibition of Notch signaling, using short hairpin RNA (shRNA) targeting Notch1, reduced the maximum population doublings of human umbilical vein endothelial cells (HUVEC), increased the activity of senescence-associated beta-galactosidase, and up-regulated the expression of aging-associated molecules such as p53, p21, and p16. Knockdown of the Notch ligand Jagged1 resulted in attenuation of Notch signaling, thereby inducing premature senescence in a way similar to knockdown of Notch1. In contrast, over-expression of Notch1 or Jagged1 extended the replicative lifespan of HUVEC and decreased the expression of aging-associated molecules. To elucidate the mechanism how inhibition of Notch signaling induces premature senescence in endothelial cells, we examined expression of various molecules by microarray analysis, and found that the expression of Id1 (inhibitor of DNA binding 1) and MKP1 (MAP kinase phosphatase 1) was significantly down-regulated by knockdown of Notch. Over-expression of Id1 improved Notch1 inhibition-induced premature senescence, which is associated with a decrease of p16 expression. Likewise, treatment with SB203580, an inhibitor of p38 MAPK, extended lifespan of Notch-deleted endothelial cells along with down-regulation of p16. These results suggest that Notch signaling and the downstream molecules (Id1 and p38) regulate endothelial cell senescence presumably via a p16-dependent pathway, and may be a new target of the therapy for age-associated vascular diseases.
Abstract 121: GRK5-NT Regulates the Activity of Calcium-Calmodulin-Dependent Transcription Factors
Authors: Sorriento Daniela , Fusco Anna , Santulli Gaetano , Trimarco Bruno , Iaccarino Guido
Published: July 2021

We have demonstrated that the amino terminal domain (GRK5-NT), which includes the RH domain and the region of binding to calmodulin, is able to regulate cardiac hypertrophy through inhibition of NFκB. Several studies indicate that mechanical stress and neuro-hormonal activation (Angiotensin, phenylephrine, endothelin) increase the levels of intracellular calcium, therefore activating calcineurin, a serine/threonine phosphatase that dephosphorylates nuclear factor of activated T cells (NFAT). Aim of this study is to evaluate the possibility that GRK5-NT regulates calcium-calmodulin dependent transcription factors. To this aim, we infected cardiomyoblasts in culture with an adenovirus encoding for GRK5-NT (AdGRK5-NT) or treated with a synthetic protein (TAT-RH), which reproduce the only RH domain of GRK5. Hypertrophic responses were induced by chronic stimulation of α
1
adrenergic receptors with phenylephrine (PE 10
-7
M,24h) and the levels of GATA4 and NFAT3 were assessed by western blot. PE induces activation and nuclear translocation of GATA4 and NFAT3 (NFAT:+38±3%;GATA4:+46±3% vs control), the treatment with AdGRK5-NT, but TAT-RH, reduces this effect (NFAT:-68±5%;GATA4:-56±2% vs PE). These data suggest that GRK5-NT using the NT domain regulates the activation of calcium-calmodulin dependent transcription factors through a mechanism of competition for binding to calmodulin. To confirm these data, we evaluated the effect of GRK5-NT
in vivo
in spontaneously hypertensive and hypertrophic rats (SHR). The overexpression of GRK5-NT in the heart was induced by intracardiac injection of 10
10
pfu/ml of AdGRK5-NT. The treatment inhibits nuclear translocation of NFAT3 and GATA4 (NFAT:-55±1%;GATA4:-34±3% vs PE) and is associated with a reduction in their DNA binding activity, as assessed by EMSA (NFAT:-47±1.5%;GATA4:-33±1% vs EP). In conclusion, our data indicate that GRK5-NT is able to regulate cardiac hypertrophy in two ways: inhibition of NFκB through binding and stabilization of IκB nuclear and inhibition activity of calcium- calmodulin dependent transcription factors, possibly by competing with calmodulin binding.
Abstract 124: Fetal-Like RNA Splicing Remodeling in Failing Heart Revealed by Total Transcriptome Analysis
Authors: Gao Chen , Ren Vincent , Xiao Grace (Xinshu) , Chen Jaunian , Wang Yibin
Published: July 2021

The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is also implicated in many human diseases including cancer. However, little knowledge is available about the scope of alternative splicing at whole genome level in heart diseases and even less about the mechanisms underlying the regulation of mRNA splicing in response to pathological injury in heart. Using a genome-wide RNA-Seq analysis, we have identified global alternative splicing changes associated with both development and pathological remodeling in mouse heart. Most significantly, the alternative RNA splicing events observed in failing heart mimicked the splicing profile in fetal hearts, suggesting a fetal like RNA splicing remodeling in failing hearts. After examining the expression profiles of splicing regulators in neonatal, normal adult, and failing adult hearts, Fox-1 was identified as one to be significantly down regulated in the failing and fetal hearts. Morpholino mediated Fox-1 knock-down in zebrafish embryos led to lethal phenotype associated with impaired cardiac development and function. This phenotype could be rescued by re-expressing both zebrafish and mouse Fox1 gene. Therefore, our established functional significance of Fox1 mediated RNA alternative splicing serves as a key molecular player in transcriptome remodeling during cardiac development and pathology.
Abstract 13: Nur77 Translocation from Nucleus to Mitochondria Contributes to the Enhanced Susceptibility to Ischemia-Reperfusion Injury in Diabetic Hearts
Authors: Liu Jingyi , Liu Peilin , Jia Min , Zhang Haifeng , Gao Chao , Yan Wenjun , Liu Yi , Yang Lu , Sun Lu , Tao Ling
Published: July 2021

Diabetes is associated with increased prevalence of ischemic heart disease which accounts for the high incidence of death in the diabetic population. In response to various pro-apoptotic treatments, Nur77, a nuclear orphan receptor, translocates from the nucleus to mitochondria where it induces cytochrome c release and apoptosis in cancer cells. However, the role of Nur77 in diabetic myocardial ischemia/reperfusion (MI/R) injury remains elusive. Ten-week-old male C57BL/6J mice (WT) and ob/ob mice (OB) were subjected to 30 min of ischemia and 3 or 24 hrs of reperfusion. Compared with WT, the OB hearts exhibited more severe MI/R injury as evidenced by more ROS production, increased infarct size, caspase-3 activity, TUNEL staining and decreased cardiac function (n=8, all
P
<0.05). There was no significantly difference in total Nur77 expression levels between sham and MI/R group in WT mice. However, MI/R caused significant Nur77 mitochondria translocation as evidenced by decreased nuclear and increased cytosolic/mitochondrial Nur77 levels (all
P
<0.01). Comparing to WT, total Nur77 expression levels were significantly reduced in OB heart before MI/R (
P
<0.01 vs. WT). However, MI/R caused more significant Nur77 mitochondrial translocation in OB mice, reaching a level that was not different from that seen in WT after MI/R (
P
>0.01 vs. WT). Intramyocardial small-interfering RNA (Nur77-siRNA) injection specifically knocked-down the myocardial Nur77, increased ejection fraction and reduced infarct size/apoptosis after MI/R in both WT and OB mice (all
P
<0.05). However, more significant cardioprotection of Nur77 knockdown was observed in OB mice than that seen in WT. Collectively, we demonstrated firstly that the increased translocation of Nur77 from nucleus to mitochondria partly contributes to the enhanced susceptibility to MI/R in diabetic hearts. Interventions blocking this subcellular redistribution of Nur77 may be a potential therapy for the treatment of diabetic heart injury.

Key Words:
Nur77 • Mitochondria Translocation • Myocardial ischemia/reperfusion • Diabetes
Abstract 130: Il-10 Regulates the Immunoregulatory Response in the Regression of Atherosclerosis
Authors: McCarthy Cathal , Duffy Michelle , Mooney Declan , James William , Fitzgerald Desmond J , Griffin Matthew D , Belton Orina
Published: July 2021

We have previously shown that dietary administration of conjugated linoleic acid (CLA) induces regression of pre-established atherosclerosis in the apoE
-/-
mouse,
via
modification of inflammatory cell function. However, the exact mechanism through which this occurs has not been elucidated. Here we describe a functional role for signaling of the anti-inflammatory cytokine, IL-10, in atherosclerosis regression and investigate the consequence of enhanced IL-10 generation on the immune cell population
in vivo
.

Our initial aim was todelineate the atheroprotective mechanisms modulated by CLA. Transcriptomic analysis of aortic tissue in the CLA-induced regression model identified an enrichment of the IL-10 signaling pathway. Further analysis of the network identified increased IL-10 receptor expression (localized to the macrophage cells) and STAT3 phosphorylation; and increased expression of downstream target genes such as the anti-inflammatory cytokine IL-1Ra (by 3.45 ± 0.83 p<0.05 fold) and suppressor of cytokine signaling (SOCS3) (by 2.24± 0.44 p<0.01 fold). Moreover, there was increased circulating IL-10 serum levels in apoE
-/-
mice fed a CLA supplemented 1% cholesterol diet compared with apoE
-/-
mice fed a 1% cholesterol diet alone (41.9 ± 8.9 vs 79.8 pg/ml ± 22.4 p<0.01). Interestingly, both IL-10 production and STAT3 phosphorylation was significantly increased in bone marrow derived macrophages from CLA fed mice.

We next employed flow cytometry to delineate the phenotype of single cell suspensions of aortae. CLA supplementation regulated immune cell infiltration of the aorta with increased number of the anti-inflammatory Ly6C
lo
monocytes evident during regression (29±8 vs 77±14cells/mg aorta p<0.05). In addition, CLA-induced regression was associated with increased polarization towards an anti-inflammatory M2 phenotype, confirmed by an enrichment of M2 genes in the aorta, which occurred as a consequence of increased aortic IL-10 production. In summary CLA mediated induction of IL-10 signaling alters the immunoinflammatory response to atherosclerosis, with increased volume of Ly6C
lo
monocytes infiltrating the regressing lesions and directed polarization of macrophages towards an M2 phenotype in the plaque microenvironment.
Abstract 138: Procollagen I Carboxyterminal Propeptide Levels in Friedreich Ataxia and Its Relation to Subclinical Myocardial Disease
Authors: Chacko Paul , Jin James , Tran Tam , Prior Thomas W , Agarwal Gunjan , Raman Subha V
Published: July 2021

Background:
Friedreich ataxia (FA) is the most common recessive hereditary ataxia. Triplet expansion (GAA) of the frataxin gene leads to intramitochondrial iron accumulation, and cardiomyopathy is the leading cause of death in FA. Histopathologic analysis of autopsy hearts has shown myocardial fibrosis and concentric left ventricular (LV) thickening, which our group has previously shown in vivo in FA patients using cardiac magnetic resonance with late gadolinium enhancement (LGE-CMR). Procollagen I carboxyterminal propeptide (PICP) is a serum marker of collagen synthesis, suggesting potential utility as a biomarker of myocardial fibrosis and remodeling. Objectives: The aims of this work were to 1) determine PICP levels in FA patients compared to similar-aged healthy controls, 2) evaluate PICP levels relative to GAA repeat length, 3) evaluate PICP in the context of cardiac remodeling and 4) compare serum PICP levels to presence or absence of fibrosis by LGE-CMR.

Methods:
30 FA patients (34.2±11.8 years) underwent measurement of serum PICP using sandwich ELISA as well as CMR examination with cine and LGE acquisitions. Southern blot analysis was performed to measure GAA repeats, recording the minimum of 2 values for each FA patient. 5 healthy volunteers (37.6±8.4 years) served as controls for PICP measurement. Cine images were analyzed at end-diastole to compute relative wall thickness (RWT), a marker of concentric remodeling.

Results:
PICP values were markedly elevated in FA patients compared to normal controls (1034±76 vs. 657±89 ng/mL, p=0.004). Minimum GAA repeat length was moderately correlated with greater RWT (r=0.40, p=0.041) but did not predict PICP level. Higher PICP level modestly predicted worse concentric remodeling (r=0.39, p=0.033). However, PICP did not distinguish between 15 patients with evident myocardial fibrosis by LGE vs. 15 LGE-negative FA patients (1042±128 vs 1027±87 ng/mL, p=0.925).

Conclusions:
PICP, a serum marker of collagen synthesis, is significantly elevated in FA patients with higher values indicating adverse LV remodeling. PICP may provide complementary information to direct myocardial fibrosis imaging, and warrants further evaluation as a biomarker of disease severity and treatment response.
Abstract 139: Ultrasound Stimulation Restores Impaired Neovascularization-Related Capacities of Human Circulating Angiogenic Cells
Authors: Toyama Yasuyuki , Sasaki Ken-ichiro , Tachibana Katsuro , Ueno Takafumi , Kajimoto Hidemi , Yokoyama Shinji , Ohtsuka Msanori , Koiwaya Hiroshi , Nakayoshi Takaharu , Mitsutake Yoshiaki , Chibana Hidetoshi , Itaya Naoki , Imaizumi Tsutomu
Published: July 2021

Aims:

Unsatisfactory effects of therapeutic angiogenesis in critical limb ischemia may be ascribed to use of circulating angiogenic cells (CACs) with impaired neovascularization-related capacities of atherosclerotic patients. We tested whether ultrasound cell stimulation can restore the impaired capacities.

Methods and Results:
During four-day-culture of human peripheral blood-derived mononuclear cells to achieve CACs, we stimulated the culture cells daily with low-intensity pulsed ultrasound stimulation (LIPUS). LIPUS to healthy volunteer-derived culture cells augmented generation and migration capacities of CACs, increased concentrations of angiopoietin 2 and nitrogen oxides in the culture medium, and increased expressions of phosphorylated-Akt and endothelial nitric oxide synthetase in CACs on western blotting. LIPUS to atherosclerotic patient-derived culture cells also augmented the generation and migration capacities of CACs. Although neovascularization in the ischemic hindlimb of athymic nude mice was impaired after intramuscular injection of atherosclerotic patient-derived CACs compared with that of healthy volunteer-derived CACs, LIPUS to atherosclerotic patient-derived culture cells restored the impaired neovascularization.

Conclusion:
Therapeutic angiogenesis with LIPUS-pretreated CACs may be a new strategy to rescue critical limb ischemia in atherosclerotic patients.
Abstract 140: Effect of Acute β-Adrenergic Receptor Stimulation on Cardiac Function in Gravin Knockout Mice
Authors: Guillory Ashley N , Wijaya Cori S , Yin Xing , Diaz Diaz Andrea , Rababa'h Abeer , McConnell Bradley K
Published: July 2021

Gravin, an A kinase anchoring protein expressed in the myocardium, localizes protein kinase A (PKA), protein kinase C and other signaling molecules to the β2-adrenergic receptor (β2-AR). PKA modulates cardiac function by phosphorylating key substrates of the excitation-contraction coupling mechanism such as troponin I, phospholamban, L-type Ca2+ channels and the ryanodine receptor. PKA is activated upon agonist stimulation of β-ARs, thus, the purpose of this study was to determine whether disruption of the PKA/Gravin interaction would alter cardiac function upon β-AR stimulation. WT and gravin-knockout (KO) mice were given an acute challenge with isoproterenol (ISO; 0.25μg/g) injected intraperitoneally, then cardiac function was measured via echocardiography. Control animals were injected with an equal volume of 0.002% ascorbic acid. All experiments were performed with n≥6. Baseline electrocardiography revealed no differences between the WT and KO mice prior to treatment. Additionally, there were no significant differences in body weight or left ventricular (LV) mass between the WT and gravin-KO mice. As expected, heart rate was equally increased in both ISO treated groups when compared to their respective controls (WT vehicle: 489±28.9; KO vehicle: 447.8±20.1; WT ISO: 596.4±8.1; KO ISO: 570.7±16.4; BPM; p<0.0001). However, cardiac output was significantly increased in the gravin-KO vehicle mice as well as in the gravin-KO mice acutely challenged with ISO compared to their WT counterparts (WT vehicle: 19.82±1.18; KO vehicle: 24.78±1.00; WT ISO: 26.97±1.35; KO ISO: 32.84±1.39; ml/min; p<0.0001). Similarly, stroke volume was also much greater in both the vehicle and ISO treated gravin-KO mice than the respective WT group (WT vehicle: 43.94±4.01; KO vehicle: 51.30±2.36; WT ISO: 54.47±4.16; KO ISO: 68.03±2.67; µl; p=0.0056). Finally, LV fractional shortening was also significantly enhanced in both gravin-KO groups when compared with WT mice (WT vehicle: 33.89±2.47; KO vehicle: 43.00±3.25; WT ISO: 54.47±4.16; KO ISO: 68.03±2.67; %; p<0.0001). This data indicates that disruption of gravin’s scaffold mediated signaling is able to increase baseline contractility as well as augment contractility in response to acute β-AR stimulation.
Abstract 141: Bicuspid Aortic Valve Hemodynamic Abnormalities Promote Early Development of Calcific Aortic Valve Disease
Authors: Sun Ling , Chandra Santanu , Sucosky Philippe
Published: July 2021

INTRODUCTION:
The bicuspid aortic valve (BAV) is the most common congenital cardiac anomaly and is frequently associated with calcific aortic valve disease (CAVD). Although CAVD also develops in the normal tricuspid aortic valve (TAV), its progression in the BAV is more rapid. While the accelerated calcification of BAV leaflets has been linked to genetic and atherogenic predispositions, hemodynamic abnormalities are increasingly pointed as potential pathogenic contributors.

HYPOTHESIS:
Supported by our previous work, which demonstrated the sensitivity of valve leaflets to the surrounding blood flow and associated wall-shear stress (WSS), we hypothesize that the abnormal WSS experienced by BAV leaflets contribute to CAVD development by promoting valvular inflammation, remodeling and osteogenic differentiation.

OBJECTIVE:
This study aims at comparing
ex vivo
the effects of TAV and BAV leaflet WSS on valvular pathogenesis.

METHODS:
The native, side-specific WSS experienced by TAV and type-I (i.e., fused and non-coronary) BAV leaflets were obtained computationally using fluid-structure interaction simulations. Fresh porcine leaflets were subjected for 48 hours to each of the three WSS conditions using a novel double-sided shear stress bioreactor. Tissue response was characterized via Western blot and immunohistochemistry in terms of markers of endothelial activation (VCAM-1, ICAM-1), paracrine expression (BMP-4), TGF-β/Wnt signaling pathways (TGF-β1, β-catenin), extracellular matrix remodeling (cathepsin L, MMP-2, MMP-9) and osteogenic differentiation (α-SMA, osteocalcin).

RESULTS:
No significant differences in VCAM-1 and ICAM-1 expressions were detected between tissue exposed to TAV and BAV WSS. While the native WSS experienced by the TAV and non-coronary BAV leaflets maintained tissue homeostasis, tissue exposure to the fused BAV leaflet WSS resulted in a significant pathological response marked by the upregulations of BMP-4, β-catenin, MMP-2 and osteocalcin expressions.

CONCLUSION:
This study demonstrates the pathological nature of the native BAV hemodynamics and confirms the higher susceptibility of the fused BAV leaflet to calcify. The results provide new insights into the hemodynamic theory of BAV calcification.