bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3885: Adenosine A
2A
and A
2B
Receptors Work in Concert to Produce a Strong Protection against Reperfusion Injury by Targeting the mPTP via a GSK-3β-Dependent Mechanism
Authors: Xi Jinkun , Lee SungRyul , Zhu Min , McIntosh Rachel , Mueller Robert A , Norfleet Edward A , Xu Zhelong
Published: July 2021

While the role of adenosine A
2
receptor activation in modulation of reperfusion injury has been extensively investigated, controversies still exist regarding its exact role (protective or not). We aimed to test if a simultaneous stimulation of A
2A
and A
2B
receptors can produce a strong protection against reperfusion injury by targeting mitochondrial permeability transition pore (mPTP) and glycogen synthase kinase 3β (GSK-3β). NECA (100 nM) given at reperfusion reduced infarct size in isolated rat hearts (14.1 ± 1.9 % vs. 37.9 ± 3.1 % of risk zone in control), an effect that was blocked by both the A
2A
antagonist
SCH58261
(36.6 ± 2.4 %) and the A
2B
antagonist MRS1706 (39.2 ± 2.9 %), indicating that both the receptor subtypes are involved in the anti-infarct effect of NECA. NECA increased GSK-3β phosphorylation at Ser
9
and translocated GSK-3β to mitochondria upon reperfusion, an effect that was blocked by both
SCH58261
and MRS1706, implying that NECA may protect the heart at reperfusion by targeting GSK-3β and mitochondria through activation of both A
2A
and A
2B
receptors. In support of this interpretation, NECA prevented oxidant-induced loss of mitochondrial membrane potential (MMP) in cardiomyocytes (91.2 ± 3.7 % vs. 38.8 ± 5.4 % in control), which was also partially blocked by both
SCH58261
and MRS1706, indicating that NECA prevents mPTP opening by stimulating both A
2A
and A
2B
receptors. Further experiments showed that a combination of
CGS21680
(A
2A
agonist) and BAY60 – 6583 (A
2B
agonist) induced a greater protection against the loss of MMP (84.7 ± 1.1 %) than that induced by
CGS21680
(55.6 ± 2.9 %) or BAY60 – 6583 (63.7 ± 5.0) alone, corroborating the above observations. In conclusion, these data suggest that A
2A
and A
2B
receptors work in concert to prevent reperfusion injury by targeting mPTP through a GSK-3β-dependent mechanism.
Abstract 3893: A Novel IKK Inhibitor IMD-0560: Suppresses Chronic Remodeling after Myocardial Ischemia
Authors: Suzuki Jun-ichi , Wakatsuki Shin , Ogawa Masahito , Muto Susumu , Itai Akiko , Isobe Mitsuaki
Published: July 2021

Amplification of inflammatory response in the non-infarct area plays an important role in the pathogenesis of ventricular remodeling after myocardial ischemia. Activation of nuclear factor-kappa B (NF-kB) is involved in this amplification through a positive feedback loop of proinflammatory cytokines. We investigated the efficiency of IKK blockade with IMD-0560, a novel inhibitor of IKK, in a rat myocardial ischemia model. Left coronary artery occlusion (28 days) was carried out in Sprague-Dawley rats. The rats were assigned randomly to the two groups:

IMD-0560 injection (5mg/kg i.p.) daily for 28 days (MI + IMD group, n=10);

vehicle injection (i.p.) daily for 28 days (MI + vehicle group, n=10); and

thoracotomy with LAD isolation without ligation (Sham group, n=5).

Treatment with IMD-0560 significantly improved cardiac function, as indicated by the preservation of % fractional shortening (vehicle-treated, 20.2±1.5; IMD-0560-treated, 26.7±2.4; P < 0.05), and lower serum brain natriuretic peptide level (vehicle, 0.079±0.040; IMD-0560, 0.033±0.016; P < 0.05). Histological analysis showed that hypertrophy of myocardium was suppressed in the peri-infarct area (vehicle, 756.71±48.57mm2; IMD-0560, 633.35±29.83mm2; P < 0.05), and the thickness of infarct area was maintained (vehicle, 0.890±0.027mm; IMD-0560, 1.136±0.087mm; P < 0.05). The sham group showed no effect. Moreover, in situ zymography showed that matrix metalloproteinase-9 activity was inhibited in the infarct area. We revealed that IMD-0560 is potent in the suppression of chronic ventricular remodeling after myocardial ischemia.
Abstract 3905: MSCs
Wnt11
Derived Paracrine Factors Are Critical to the Repair of Infarcted Myocardium
Authors: Zuo Shi , Dai Ying , Ning Xu , Wang Yigang , Ashraf Muhammad , Xu Meifeng
Published: July 2021

Genetically engineered MSCs have been shown to serve as an effective vehicle for the delivery of protective proteins. We hypothesized that Wnt11 enhanced MSCs induced cardioprotection of ischemic myocardium via paracrine effect. We have successfully transfected Wnt11 into MSCs.

Method: Conditioned medium (con-M) was obtained from various MSCs which were exposed to normoxia or hypoxia for 24 hours and was concentrated 100 times by ultrafiltration. Con-M (50μl) was injected into the border zone of infarcted myocardium immediately after rat underwent left anterior descending coronary artery (LAD) occlusion. Cultured cardiomyocytes (CM) were also treated with Con-M before cells were exposed to hypoxia.

Result: A marked improvement in cardiac function was observed in the con-M from MSCs
Wnt11
compared to the con-M from MSCs
GFP
(Table 1

). Microarray analysis revealed that Wnt11 was 204 fold higher in MSCs
Wnt11
than that in MSCs
GFP
. Moreover, many growth factors and cytokines were upregulated when MSCs
Wnt11
were exposed to hypoxia for 24 hours (e.g. BMP4, 1.64; FGF1, 1.61 and TGFμ2, 2.21 fold higher than that in MSCs
GFP
, respectively; all p<0.05). Con-M significantly improved the survival of CM as expressed by an increased intake of MTT when CM were exposed to hypoxia. Neutralizing antibodies, including anti-Wnt11 (6 and 12 μg/ml) (Fig. 1

), anti-BMP4 (3μg/ml), anti-FGF-1 (6μg/ml) and anti-TGFβ-2 (12μg/ml), abrogated the protection of CM induced by con-M, especially anti-FGF-1 (Fig. 2

). It is concluded that MSCs
Wnt11
protect cardiomyocytes by releasing cytoprotective factors in their environment.

Table 1.

Effects of condition medium on cardiac function

Figure 1.

Anti-Wnt11 antibody increased CM injury and abrogated the protective effect of conditioned medium against hypoxic damage. N = 4 in each group

Figure 2.

Neutralizing antibodies abolished the CM protection on cultured CM. #, p<0.05
vs
normal control; *, p<0.05
vs
hypoxia; †, p<0.05
vs
Con-M-MSC
GFP
, ‡, p<0.05
vs
Con-M-MSC
Wnt11
respectively.
Abstract 3915: Del-1, A New β2-Integrin Ligand, Which Inhibits Adhesion and Homing of Progenitor Cells
Authors: Chavakis Emmanouil , Hain Andreas , Orlandi Alessia , Carmona Guillaume , Quertermous Thomas , Chavakis Triantafyllos , Dimmeler Stefanie
Published: July 2021

Progenitor cells (PC) are recruited to ischemic tissues and improve neovascularization. Beta2-integrins are essential for adhesion, transmigration and homing of PC to ischemic tissues. Developmental Endothelial Locus-1 (Del-1) is an extracellular matrix protein, that binds alphaVbeta3- and alphaVbeta5-integrins and is up-regulated during ischemia. Therefore, we investigated the role of endogenous Del-1 for angiogenesis and homing functions of PC. The Del-1-deficient mice (Del-1
−/−
) displayed a significantly increased angiogenic response in ischemic muscles in comparison to the wild type (WT) mice in the model of hind limb ischemia. However, when we assessed the role of Del-1 in HUVEC
in vitro
, silencing of Del-1 by siRNA did not affect angiogenic sprouting. Moreover, the ischemic muscles of Del-1
−/−
mice displayed a higher infiltration with CD45
+
hematopoietic cells than WT mice, suggesting that Del-1 may have an inhibitory effect on homing of PC and inflammatory cells to ischemic tissues. Interestingly, in adhesion assays human endothelial progenitor cells (EPC) and murine Lin
−
progenitor cells bound to Del-1 via beta2-integrins, but not via the alphaVbeta3- and alphaVbeta5- integrins. Furthermore, soluble Del-1 significantly inhibited the adhesion of EPC to HUVEC monolayers and to the major beta2-integrin-ligand, ICAM-1, raising the possibility that Del-1 is a beta2-integrin-inhibitor. Indeed, WT murine bone marrow mononuclear cells displayed higher adhesion rates on Del-1-deficient murine lung endothelial cells (LEC) than on WT LEC. In order to investigate the role of Del-1 for
in vivo
homing of PC, we intravenously injected murine fluorescence-labeled WT Lin
−
bone marrow PC in WT and Del-1
−/−
mice 2 days after the induction of hind limb ischemia. Interestingly, the homing of injected Lin
−
cells to ischemic muscles was significantly increased in Del-1
−/−
in comparison to WT mice (200± 30 % increase). Taken together, endogenous Del-1 is a new beta2-integrin ligand, which blocks beta2-integrin-dependent adhesion and homing of PC to ischemic tissues. It is conceivable, that endogenous Del-1 may reduce ischemia-induced neovascularization through an inhibitory effect on the beta2-integrins of progenitor and inflammatory cells.
Abstract 3942: Identification Of Endothelin Type-A Receptor-expressing Cells Involved In Embryonic Brain Angiogenesis
Authors: Ko Toshiyuki , Nishiyama Koichi , Koseki Hiroaki , Hasegawa Sho , Sato Takahiro , Asai Rieko , Kawamura Yumiko , Kurihara Yukiko , Kurihara Hiroki
Published: July 2021

Interaction between endothelial cells (ECs) and mural cells (pericytes/smooth muscle cells) is crucial for vascular stabilization and maturation during the late phase of angiogenesis. However, underlying mechanisms and its role in the early sprouting angiogenesis remain largely unveiled. Recently, we have established mice in which EGFP is knocked-in into the endothelin type-A receptor (EdnrA) locus by Cre recombinase-mediated cassette exchange method (RMCE), resulting in the visualization of EdnrA-expressing cells including neural crest (NC)-derived ectomesenchymal cells. We found sporadic distribution of EGFP+cells also in the neural tube (NT) after E9.5, when sprouting angiogenesis into the brain starts. We then examined possible involvement of these cells in angiogenesis. EGFP+cells were first observed between E9.5 to E10.0 in the NT close to its outer surface. Thereafter, the cells tended to distribute toward the ventricular zone. Interestingly, most EGFP+cells in the NT were associated with forming vessels marked by CD31. At E9.5, when migrating CD31+cells were penetrating into the NT, many sprouts were accompanied by EGFP+cells. EGFP+cells expressed PDGFRβ, a marker for mesenchyme-derived mural cells, whereas the cells were negative for CD31, PDGFRα, GFAP, nestin and Iso-B4-lectin binding. At the late gestation stage (E18.5), EGFP+cells formed a layer underlying ECs and became partly positive for α-smooth muscle actin. These results are consistent with the previous finding that mural cells of the forebrain blood vessels originate from the NC, and indicate that CD31+EC/angioblasts and EGFP+immature cells may coordinately give rise to vascular networks in the brain from the early angiogenic stage. It is also noteworthy that EGFP+cells were closely associated with angiogenic ECs in aortic ring assay and in vivo ischemic lesion in the mouse hindlimb. Although these EGFP+cells are not apparently of NC origin, the identification of EdnrA-expressing cells may clarify mechanisms for mural cell differentiation and cell-to-cell interaction involved in angiogenesis. Furthermore, the RMCE knock-in system enables us to unveil the secrets of mural cells in angiogenesis by various genetic modifications.
Abstract 3944: Converged Signaling of β-Catenin and Notch Induces Differentiation of Arterial Endothelial Cells from VEGFR2+ Vascular Progenitors
Authors: Yamamizu Kohei , Yamashita Jun K
Published: July 2021

We have been investigating molecular mechanisms of vascular development and diversification using embryonic stem (ES) cells. Previously, we established an ES cell differentiation system that reproduces early vascular development using vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2)-positive cells as common vascular progenitors (Nature, 2000). Endothelial cells (ECs) were induced from VEGFR2
+
progenitor cells with VEGF. VEGF alone mainly induced venous ECs, which are negative for arterial EC markers, ephrinB2 and CXCR4. Interestingly, addition of 8bromo-cAMP with VEGF activated Notch signaling in ECs and substantially induced arterial ECs. Nevertheless, activation of Notch using notch intracellular domain (NICD)-estrogen receptor fusion protein failed to induce arterial ECs (Arterioscler Thromb Vasc Biol, 2006). These results indicate that Notch activation was not sufficient to induce arterial ECs from vascular progenitors. Then we further investigated the downstream of cAMP, which would suffice signals for constructive induction of arterial ECs. We found that phosphatidylinositol-3 kinase (PI3K) inhibitor, LY294002, completely inhibited cAMP-induced arterial EC differentiation as well as Notch activation. GSK3β inhibitor, Bio, partially reversed the inhibitory effects of LY294002. Whereas activation of β-catenin signaling alone using Doxycycline-induced constitutive active form of β-catenin expression showed only a weak effect on arterial EC induction, simultaneous activation of β-catenin and Notch signaling completely restored arterial EC induction even in the absence of cAMP. Thus, cAMP-PI3K-GSK3β-β-catenin pathway plays a critical role in arterial EC differentiation. Furthermore, chromatin immunoprecipitation assay on the two RBP-J binding sites of mouse EphrinB2 revealed that RBP-J, NICD, and β-catenin formed a protein complex on these two sites only in arterial ECs, but not in venous ECs. These findings suggest that Notch and β-catenin signaling converged to form a novel arterial EC-specific protein complex to induce arterial ECs. This study would provide novel understandings of cellular and molecular mechanisms for arterial-venous specification and novel molecular targets for drug discovery.
Abstract 3955: Chemokines Fused to a Fractalkine Backbone and a GPI-Anchor Attract Embryonic Endothelial Progenitor Cells to the Site of Ischemia
Authors: El-Aouni Chiraz , Globisch Franziska , Pfosser Achim , Stachel Georg , Hinkel Rabea , Boekstegers Peter , Nelson Peter , Kupatt Christian
Published: July 2021

Recruitment of endothelial progenitor cells to the sites of ischemia is a prerequisite for efficient therapeutic neovascularization via vasculogenesis. Chemokines play a major role in the homing of EPCs at the ischemic vasculature, a mechanism fading in chronic ischemia. To overcome this limitation, we constructed an artificial adhesion molecule consisting of a GPI-anchor, a fractalkine-backbone and an SDF-1 head (SDF-1-fra-GPI), which was applied for enhanced recruitment of embryonic EPCs (eEPCs: CXCR4++, Tie2++, Thrombomodulin++, CD34-, MHCI-, vWF inducible, eNOS inducible)
in vitro
and
in vivo
.

Methods:
In a flow chamber adhesion assay, Control plasmids (pcDNA or GPI-SDF-1 cDNA) were compared to the SDF-1-fra-GPI construct for eEPC recruitment 24h after liposomal transfection of rat endothelial cells. In vivo, in rabbits (n=5 per group) at day 7 (d7) after femoral artery excision, 1 mg of the SDF-1-fra-GPI or eGFP cDNA was transfected into the ischemic limb. At d9, ischemic hindlimbs were retroinfused with 5x10
6
eEPCs. Angiography was performed for collateral quantification and frame count score at d9 and d37 (% of d9), capillary density was assessed via PECAM-1-staining (capillaries/muscle fiber = c/mf).

Results:
In vitro, eEPC adhesion (16±12 cells/field) was increased to a higher extent by SDF-1-fra-GPI (79±13) than SDF1-GPI (54±8) or control vector (37±8).
In vivo
, eEPC adhesion in the ischemic hindlimb after SDF-1-fra-GPI transfection compared to mock transfection (30±3 vs. 9±1 cells/field). Whereas capillary density was unaffected (1.66±0.30 SDF-1-Fra-GPI vs. 1.56±0.29 eEPCs), collateral growth (152±10% SDF-1-fra-GPI vs. 124±13%) as well as perfusion score (198±17% SDF-1-fra-GPI vs.160±6% eEPCs) further increased after SDF-1-fra-GPI transfection (controls: 1.24±0.12 c/mf, collaterals 105±8%, perfusion score 112±11%). We conclude that recruitment of EPCs expressing CXCR4 (the SDF-1 receptor) may benefit from pre-treatment of the recipient vasculature with SDF-1-Fra-GPI, an artificial adhesion molecule. This approach might be valuable for enhancing EPC recruitment in the scenario of therapeutic neovascular-ization of chronic ischemic syndromes.
Abstract 3957: RGDS-Epitope Presenting Peptide Amphiphile Nanofibers Enhance Regenerative Potency of Cell-based Strategies in Ischemic Tissue
Authors: Tongers Joern , Webber Matthew J , Renault Marie-Ange , Roncalli Jerome G , Stupp Samuel I , Losordo Douglas W
Published: July 2021

Cell-based therapies have emerged as a promising therapeutic option, however autologous approaches may be limited by the impaired potency of cells collected from patients with advanced vascular disease. Advances in bionanotechnology have led to the development of biomaterials that may offer solutions to overcome certain limitations and to enhance the therapeutic potency of cell-therapies. We evaluated a peptide amphiphile (PA) nanofiber that self-assembles 3-dimensionally, mimicing the extracellular matrix, and incorporates bioactive epitopes of interest. Based on the importance of fibronectin for endothelial progenitor cell (EPC) adhesion, motility and survival, the RGDS-epitope that functionally mimics the integrin-binding sequence of fibronectin was integrated for presentation (RGDS PA). For in vitro studies, EPCs were expanded from the bone marrow derived mononuclear cell fraction under endothelial culture conditions. Incubation with RGDS-PA (0.2w%) reduced H
2
O
2
induced apoptosis of EPCs significantly (0.20±0.02 TUNEL
+
/TUNEL
−
EPCs) compared to control cells without RGDS-PA (0.64±0.09, n= 5–7, P<0.01). RGDS-PA also induced proliferation of EPC in vitro (MTS assay; RGDS-PA 356±42% vs. 100% control, n= 5–9, P<0.01). Pre-incubation/Culture of EPCs with RGDS-PA led to an increase in adherent cells on uncoated (32±10 vs. 3±1, P<0.05), vitronectin (130±18 vs. 5±3, P<0.01) and fibronectin (109±12 vs. 39±2, P<0.05) coated but not on collagen coated surfaces compared to control cells. Hind-limb ischemia was surgically induced in 8 week-old, male FVB/N mice by complete ligation of the proximal femoral artery including all superficial and deep branches; postoperatively ischemia was verified by laser Doppler imaging. Suspension of
subtherapeutic
cell-doses in RGDS-PA solution for combined application enhanced recovery of laser Doppler-derived blood flow (ischemic vs. non-ischemic limb ratio: RGDS-PA 0.67±0.06 vs. saline 0.55±0.04) and reduced the amputation rate compared to cells alone at day 28 (amputation score: RGDS-PA 3.6±0.2 vs. saline 3.0±0.1; n=5– 8/group, P<0.05).In conclusion, the RGDS-epitope presenting PA nanofiber enhances survival, proliferation and adhesion of EPCs and augments potency for ischemic tissue repair.
Abstract 5251: Defective Inter-domain Interaction May Cause Spontaneous Ca
2+
Release Via Reduced Affinity of Calmodulin Binding to RyR2 in Failing Hearts
Authors: Ono Makoto , Yano Masafumi , Suetomi Takeshi , Xu Xiaojuan , Uchinoumi Hitoshi , Tateishi Hiroki , Okuda Shinichi , Kobayashi Shigeki , Yamamoto Takeshi , Ikeda Yasuhiro , Ohkusa Tomoko , Matsuzaki Masunori
Published: July 2021

We previously reported that interaction between N-terminal
1–600
and central domains
2000–2500
of ryanodine receptor (RyR2), harboring many mutation sites in CPVT, is defective (i.e. domain unzipping) in failing hearts. Here, we investigated the pathogenic role of calmodulin (CaM), one of the accessory proteins in RyR2, on Ca
2+
release in failing hearts. Sarcoplasmic reticulum (SR) vesicles were isolated from dog LV muscles {normal (N), n=6; 4-weeks rapid RV pacing (HF: n=6). To assess CaM binding to RyR2, SR was mixed with CaM-SANPAH conjugate (16nM-1μM), followed by UV photolysis. Then, the RyR2-bound CaM was detected by Western blotting using anti-CaM antibody. The affinity of CaM binding to RyR2 was lower in failing SR than normal SR (Kd: 47nM in HF: 19nM in N , p<0.01). To assess the possible relationship between domain unzipping and CaM dissociation from RyR2, RyR2 was also fluorescently labeled with methylcoumarin acetamido (MCA) using DP
2460–2495
(DPc10), which harbors a mutation site in CPVT; R2474S, as a site-directing carrier. In failing SR, domain unzipping was already taken place, as evidenced by an increased accessibility of the bound MCA to a large-size fluorescence quencher. Interestingly, addition of FK506 (10 μM), which was found to dissociate FKBP12.6 from RyR2 and to induce domain unzipping mimicking failing SR, to normal SR indeed reduced the CaM binding affinity to RyR2. In saponin-permeabilized, failing cardiomyocytes ([Ca
2+
]=buffered at 75 nM), the frequency of Ca
2+
sparks was markedly increased (SpF; s
−1
·100μm
−1
: 13.9±0.63 in HF; 7.3±0.6 in N, p<0.01). Addition of CaM (1 μM) in the presence of KN-93 (CaMKII inhibitor) inhibited the increase in SpF (7.9 ±0.41 , p<0.01). This CaM’s effect was, however, markedly inhibited by co-addition of CaM-binding domain peptide within RyR2 (3583–3603) (10.4±0.65, p=ns), strongly suggesting that the re-binding of CaM to RyR2 corrects the abnormal Ca
2+
release in failing cardiomyocytes. In conclusion, the defective inter-domain interaction between N-terminal and central domains within RyR2 seems to increase spontaneous Ca
2+
release events in failing SR, via the reduced CaM-binding to RyR2. Fixing CaM binding to RyR2 may be protective against the diastolic Ca
2+
release in failing hearts.
Abstract 5254: Dependence of Beta-3 Adrenergic Signaling on the Adipokine Leptin in Cardiac Myocytes
Authors: Larson Jill E , Yang Ronghua , Miller Karen , Phan Alexander , Barouch Lili A
Published: July 2021

Introduction:
The adipokine leptin modulates cardiac contractility via a NO-dependent mechanism. Beta-3 adrenergic receptors (B3AR) negatively regulate B-adrenergic signaling via NO and are dependent on leptin for normal expression in adipocytes, making B3AR an attractive candidate for crosstalk with leptin in the heart. Accordingly, we tested the hypothesis that cardiac B3AR signaling is dependent on leptin and is severely diminished in leptin-deficient
ob/ob
mice.

Methods and Results:
We studied fura-loaded isolated cardiac myocytes (n=11–25 cells/strain) from B3
−/−
,
ob/ob
, and their respective background strains of WT mice (FVB and C57BL6) with a combined treatment of the nonspecific B-agonist isoproterenol (ISO) and the B3-specific agonist
BRL37344
(BRL). Continuous administration of ISO “primes” cells to elicit a maximal response from subsequent B3-specific stimulation. Stepwise increases in BRL produced dose-dependent reductions of sarcomere shortening and calcium transients in myocytes from both WT strains. Negative inotropic effects of BRL were absent in B3
−/−
as expected. Importantly, effects of BRL were markedly diminished in
ob/ob
myocytes. To confirm that B3AR signaling is dependent on leptin, we performed qPCR of B3AR mRNA in WT and
ob/ob
myocardial extracts. B3AR mRNA levels were indeed decreased by 78±5% (n=4–5 hearts) in
ob/ob
vs. WT, and 4 weeks of
in vivo
leptin repletion restored B3AR mRNA in
ob/ob
to WT levels.

Conclusions:
These findings demonstrate a novel role for leptin in cardiac myocyte physiology, specifically, leptin dependence of B3AR signaling. Our results offer new insight into the mechanisms of obesity-related cardiac dysfunction.
Abstract 5256: Identification of a Novel Epoxyeicosatrienoic Acid (EET) Receptor: EETs Function as Selective, Endogenous Thromboxane Receptor Antagonists
Authors: Behm David J , Ogbonna Andrea , Wu Charlene , Burns-Kurtis Cynthia L , Pullen Mark , Douglas Stephen A
Published: July 2021

Introduction:
Epoxyeicosatrienoic acids (EETs) are cytochrome P450 metabolites of arachidonic acid which elicit vasodilatory, anti-inflammatory and anti-thrombotic effects. Interestingly, however, the mechanism by which EETs function remains unclear and was therefore investigated in the present study. Since previous reports suggest EET mediated vasodilation is dependent on large-conductance Ca
2+
-activated K
+
(BK
Ca
) and vanilloid transient receptor potential (TRPV4) channels, the ability of 14,15 EET to induce vasodilation was investigated in BK
Ca
and TRPV4 homozygous knockout mouse isolated aorta.

Results:
Contrasting previous reports, neither BK
Ca
nor TRPV4 gene deletion significantly altered the potency of 14,15 EET-induced reversal of tone established with the thromboxane receptor [TP] agonist U46619. Surprisingly, whereas 14,15 EET reversed U46619-contractile tone of rat isolated aorta (IC
50
1.2μM), the eicosanoid had no effect on either phenylephrine- or endothelin-1-contractile tone. The remaining three EET regioisomers (5,6 EET, 8,9 EET, 11,12 EET) acted similarly, suggesting EETs might function as TP antagonists. Consistent with competitive TP antagonism, 14,15 EET pretreatment (1–10μM) inhibited U46619-induced vasoconstriction in a concentration-dependent manner (pA
2
5.9). TP antagonism was observed in mouse and dog isolated arteries and rat tertiary bronchi, indicating that the effects are not specific to one species or tissue. Accordant with the functional potency at TP, 14,15 EET bound to recombinant human TP with a K
i
of 3.2μM. In contrast, 14,15 EET showed weaker affinities for other prostanoid receptors including DP, FP, EP
1–4
and IP (K
i
s 6.1, 5.3, 42.6, 19.7, 13.2, 20.2 and >25μM, respectively) and no appreciable affinity (K
i
s >10μM) for 51 “non-prostanoid” receptors/ion channels. Consistent with these data, 14,15 EET failed to inhibit (K
b
>10μM) “non-TP” prostanoid receptor mediated function in multiple cell/tissue-based assays.

Conclusions:
EETs function as novel “endogenous” G-protein-coupled receptor competitive antagonists, inducing vasodilation via direct TP inhibition. Such observations might help develop novel strategies targeting the EET pathway for treatment of cardiovascular disease.
Abstract 5258: SDF1-Triggered Erk-2 Activation In Endothelial Progenitor Cells In Patients With Coronary Artery Disease And Healthy Controls: Role For Adhesion And Migration
Authors: Friedrich Erik , Werner Christian , Walenta Katrin , Boehm Michael , Scheller Bruno
Published: July 2021

Endothelial progenitor cells (EPCs) are a heterogeneous group of bone marrow-derived precursor cells that circulate in peripheral blood and are recruited to sites of vascular injury or tissue damage. The underlying relevant intracellular signalling cascades are poorly characterized. EPCs were isolated from healthy volunteers and from patients with severe coronary artery disease (CAD; stable coronary 3-vessel disease) by density gradient centrifugation and cultured for 4 days under endothelial selection pressure prior to stimulation with the chemokine stromal-derived factor-1 (SDF-1; 100 nM, 30sec-5min). Western blot analysis revealed not only an enhanced phosphorylation of Akt (S473), but also a selective activation of Erk-2 after 60–90sec (baseline (densitometry): 8±2, 60sec: 50±18, 90sec: 134±17 arbitrary units; AU) with a maximum after 3–5min (3min: 232±34, 5min: 232±13 AU; n=3, p<0.05). The SDF-1-triggered Erk-2-phosphorylation could be blocked by pretreatment of EPCs with the Erk-inhibitor PD98059 (10μM, 30min), the Akt-phosphorylation by the PI3-kinase-inhibitor wortmannin. In contrast, EPCs from patients with CAD were characterized by a temporally delayed and quantitatively attenuated Erk-2-phosphorylation (baseline: 1.2±0.3, maximum after 5min: 150±46 AU; n=4, p<0.05). Functionally, pretreatment of EPCs with PD98059 (10μM, 30min; n=3, p<0.05) inhibited SDF-1-triggered migration (modified Boyden chambers; control: 21901±3136, PD: 10187±886 cells/hpf) and adhesion under physiological shear stress (2 dynes/cm
2
; control: 52±4, PD: 26±3 cells/hpf). Compared to healthy controls, SDF-1-triggered dynamic adhesion of EPCs from patients with coronary 3-vessel disease was significantly diminished (control: 52±4, CAD: 21±3 cells/hpf; n=4, p<0.05). These data identify Erk-2 as a chemokine-activated signaling molecule that regulates migration and adhesion in EPCs and is down-regulated in association with functional defects in EPCs of patients with CAD. Erk-2 may be a potential novel target of EPC-based regenerative strategies for the therapy of ischemic vascular disease.
Abstract 5272: Perinatal Loss of Nkx2–5 Results in Rapid Conduction and Contraction Defects
Authors: Briggs Laura E , Takeda Morihiko , Cuadra Adolfo E , Wakimoto Hiroko , Lu Jonathan T , Yasui Kenji , Ikeda Yasuhiro , Chien Kenneth R , Kasahara Hideko
Published: July 2021

Homeobox transcription factor Nkx2–5, highly expressed in heart, is known to be a critical factor during early embryonic cardiac development. Having previously demonstrated the persistent expression of Nkx2–5 in cardiac myocytes from the precardiac mesoderm through to the adult heart, we hypothesized that Nkx2–5 actively regulates a critical set of genes in postnatal cardiomyocytes to maintain proper cardiac function. We generated tamoxifen-inducible Nkx2–5 knockout mice that carry homozygous floxed-Nkx2–5 alleles and a heterozygous Cre-ERTM transgene under the control of CMV enhancer and chicken-beta-globin promoter. A single injection of tamoxifen into pregnant mice on gestation day 19 almost completely deleted Nkx2–5 genes, as demonstrated by Southern and Northern blotting. These mice demonstrated rapid conduction and contraction defects within 4 days of tamoxifen-injection. Conduction defect was accompanied by reduction of the cardiac voltage-gated Na+ channel pore-forming a-subunit (Nav1.5-alpha) in ventricles, the largest ion channel in the heart responsive for rapid depolarization of the action potential. In addition, Ca2+ release from the sarcoplasmic reticulum was diminished accompanied by substantial reduction in ryanodine receptor 2. Because excitation and contraction is tightly coupled in hearts, reduced handling of Na+ and Ca2+ demonstrated in Nkx2–5 knockout hearts may mutually lead to rapid conduction and contraction defects. Survival studies demonstrated that approximately 50% of Nkx2–5 knockout mice died before 3 weeks of age, and all the knockout mice that survived demonstrated conduction defects and enlarged hearts at 4 weeks of age. These defects appeared sooner and progressed more rapidly than those seen in mice with embryonic ventricular-restricted deletion of Nkx2–5, likely due to nearly complete deletion of Nkx2–5 in the entire hearts, without compensatory mechanisms during embryonic stages after loss of Nkx2–5. Our results demonstrate that Nkx2–5 is not limited to embryonic heart development; it is critical for both conduction and contraction in the perinatal heart by regulating expression of several important gene products in conduction and contraction.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 5275: Kruppel-Like Factor 15 Regulates the Cardiovascular Response to Angiotensin II
Authors: Haldar Saptarsi M , Lu Yuan , Cui Yingjie , Jeyaraj Darwin , Ibrahim Osama A , Das Manjusri , Hao Caili , Eapen Sam J , Kawanami Daiji , Doughman Yong Qiu , Watanabe Michiko , Sadoshima Junichi , Jain Mukesh K
Published: July 2021

Background
: Angiotensin II (A-II) causes pathologic remodeling of the heart and vasculature through gene-regulatory pathways that are incompletely understood. We and others have identified Kruppel-Like Factor 15 (KLF15) as a transcription factor that may exert beneficial effects in the cardiovascular system, however, its role in A-II signaling remains unknown. We hypothesize that KLF15 can modulate the deleterious effects of A-II on the heart and blood vessel.

Methods and Results
: A-II downregulates KLF15 expression in heart and vascular tissues
in vitro
and
in vivo
. Mice with systemic KLF15 deficiency (male, age 10 wks) have baseline abnormalities of the heart (increased cardiac mass, LV thickness, fetal gene expression) and vasculature (thickened aortic media with elastin breakdown). In response to chronic A-II infusion (1.4 mg/kg/D x 14D), KLF15 KO mice develop severe degenerative changes in both the heart and vessel compared to wild-type controls. In the heart, KLF15 KO mice develop pronounced heart failure characterized by augmented cardiac mass, eccentric LV remodeling, profound LV systolic dysfunction, increased fibrosis, enhanced fetal gene expression and apoptotic cell loss. Dramatic degenerative changes also occur in the aorta of KLF15 KO mice including medial destruction with elastin degradation, outward remodeling with vessel dilation, augmented inflammatory gene expression, and foci of severe smooth muscle apoptosis. Mechanistically, there is exaggerated accumulation of p53 and its direct pro-apoptotic targets in the heart (Bax) and aorta (Bax, p21) of KLF15 KO mice. As p53-mediated degradation of HIF1α has been implicated in cardiac decompensation, we show that KLF15 KO hearts have decreased HIF1α levels and fail to mount an angiogenic response to A-II. Finally, we provide evidence that KLF15 can directly inhibit p53 protein accumulation in a proteasome-dependent manner.

Conclusion
: KLF15 deficiency renders the cardiovascular system exquisitely sensitive to A-II stimulation and leads to degenerative remodeling of the heart and blood vessel with exaggerated p53 accumulation in these tissues. These findings suggest that manipulation of KLF15 function may provide novel approaches to inhibit the pathologic effects of A-II.
Abstract 5278: The Sonic Hedgehog Transcription Factor Gli3 Modulates Ischemia-induced Angiogenesis
Authors: Renault Marie-Ange , Roncalli Jerome , Tongers Joern , Misener Sol , Thorne Tina , Ito Aiko , Clarke Trevor , Millay Meredith , Kamide Christine , Klyachko Ekaterina , Losordo Douglas W
Published: July 2021

Gli transcription factors are mediators of hedgehog signaling and have been shown to be critical in several steps during development. We have shown that the Hedgehog pathway is reactivated in the adult cardiovascular system under ischemic conditions, however the specific role of Gli3 has not been elucidated. Adenoviral mediated overexpression of Gli3 promotes HUVEC migration (250±58% of control, p<0.001) while down regulation of Gli3 via siRNA delayed tube formation on Matrigel (total tube length after 8 hours 6.86 vs. 70.76 control), suggesting a possible role of Gli3 in angiogenesis. We next investigated the role of Gli3 in angiogenesis using Gli3
+/−
(Gli3
+/XtJ
) mice, a well established model of reduced Gli3 expression. VEGF-induced corneal angiogenesis was impaired in Gli3
+/−
mice compared to WT. The role of Gli3 in angiogenesis was then confirmed in two ischemia models. Hind-limb ischemia (HLI) was induced by resection of the left femoral artery. Capillary density was reduced by a mean of 48.40±12.08% in Gli3
+/−
mice vs. WT 7, 14 and 28 days. Myocardial infarction (MI) was induced by ligation of the LAD. 28 days after MI, left ventricular function assessed by echo and histological analysis revealed that Gli3
+/−
mice exhibit reduced ejection fraction (27.92±4.49% versus 37.56±7.02% for the WT, p=0.004), increased fibrosis area (33.65±9.73% versus 19.81±5.40% for the WT, p=0.007) and a decrease capillary density in the ischemic and border zones. These data indicate that Gli3 deficiency leads to impaired angiogenesis in both ischemic and non ischemic conditions. Moreover, the impairment in ischemia induced neovascularization is associated with more severe impairment of cardiac function after MI. The mechanism of Gli3’s effects was then investigated
in vitro
. Promoter reporter assays revealed that Gli3 overexpression inhibits Gli-dependent transcription, while Western analysis show
increased
Akt phosphorylation, activation of the ERK1/2 and increased c-Fos expression. Using a dominant negative Akt expressing virus and a MEK1/2 inhibitor, we show that Gli3 induced-EC migration is dependent on Akt and ERK1/2. These studies provide the first evidence that the Gli3 transcription factor regulates angiogenesis and EC phenotype.
Abstract 5279: MCPIP1 is an Inducible Corepressor of NF-KappaB
Authors: Liang Jian , Kolattukudy Pappachan E , Fu Mingui
Published: July 2021

The transcription factor NF-κB is a central regulator of host immune and inflammatory response. However, excessive and prolonged activation of NF-κB can cause massive damage to host tissues and can result in human inflammatory diseases, such as atherosclerosis and arthritis. Thus, NF-κB activation must be terminated at the appropriate time points. MCPIP1 is a recent identified CCCH-type zinc finger containing protein, which is significantly induced by MCP-1 treatment of human monocytes and thus designated as MCP-induced protein1 (MCPIP1). Here we report that MCPIP1 function as an inducible corepressor of NF-κB to feedback control NF-κB activation. In the transfection experiments, MCPIP1 dose-dependently inhibits inflammatory stimuli-induced NF-κB reporter activity. Overexpression of MCPIP1 does not affect TNFα-initiated IKK activation, NF-κB translocation and DNA binding. Further study reveals that MCPIP1 can directly bind to p65 of NF-κB and recruit NCoR and HDAC1 to form repressing complex, which blocks NF-κB target gene expression. Interestingly, the same stimuli that induce NF-κB activation also can induce MCPIP1 expression and translocation into nucleus. Taken together, our results suggest that MCPIP1 is a novel inducible corepressor of NF-κB and may be a critical player in host immune and inflammatory response.

This research has received full or partial funding support from the American Heart Association, AHA Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).
Abstract 5304: Dominant Negative Suppression of Rad Leads to Intracellular Ca
2+
Overload via Up-Regulation of Cardiac Ryanodine Receptor Activity
Authors: Murata Mitsushige , Yada Hirotaka , Yamakawa Hiroyuki , Aizawa Yoshiyasu , Yuasa Shinsuke , Hakuno Daihiko , Makino Shinji , Sano Motoaki , Murata Mitsuru , Ogawa Satoshi , Fukuda Keiichi
Published: July 2021

Background:
We have recently reported that the ras-related small G-protein Rad plays a critical role in generating arrhythmias via regulation of L-type Ca
2+
channel. However, it has remained unclear whether or not the mechanism for its arrhythmogenesis is attributed only to L-type Ca
2+
channel activity. This study was designed to demonstrate the role of Rad in intracellular calcium homeostasis by cardiac-specific dominant negative suppression of Rad.

Methods and results:
Transgenic mice that overexpress dominant negative mutant Rad (DN Rad TG) driven by α-myosin heavy chain promoter were generated. To measure intracellular Ca
2+
concentration ([Ca
2+
]
i
), we recorded [Ca
2+
]
i
transients and Ca
2+
sparks from isolated cardiomyocytes by confocal microscope. The mean amplitude of [Ca
2+
]
i
transient was significantly increased in DN Rad TG cardiomyocytes, compared with WT littermate mouse cells (F/F
0
3.3 ± 0.2, n = 25, in DN Rad TG cells vs. 2.4 ± 0.1, n = 30, in WT cells, P<0.05). The frequency of Ca
2+
sparks was significantly higher in TG cells than in WT cells (5.2 ± 0.6 sparks•100 μm
−1
•s
−1
, n = 45 in TG cells vs. 1.9 ± 0.3 sparks•100 μm
−1
•s
−1
, n = 45, in WT controls, P<0.05), although there were no significant differences in their amplitudes. Furthermore, SR Ca
2+
content was not altered in TG cells, as assessed by caffeine-induced [Ca
2+
]
i
transient. These results suggested that the properties of fundamental SR Ca
2+
release units might have been affected by the inhibition of endogenous Rad activity. To elucidate the mechanism for the increased frequency of SR Ca
2+
release channel, phosphorylation of Ser
2809
on cardiac ryanodine receptor (RYR2) was examined. The phosphorylation of RYR2 at Ser
2809
was significantly enhanced in TG mouse hearts compared with WT mice, implicating the upregulation of RYR2 activity in TG mice. Additionally, this Rad-mediated phosphorylation of RYR2 was regulated by protein kinase A (PKA) activity.

Conclusions:
Our results provided the first evidence that Rad regulated RYR2 activity via PKA signaling pathway. Thus, Rad might play a critical role in cellular Ca
2+
homeostasis via its inhibitory effects on RYR2 as well as L-type Ca
2+
channel.
Abstract 5316: hERG 1b as a Potential Target for Inherited and Acquired Long QT Syndrome
Authors: Robertson Gail A , Sale Harinath , Tester David , O’Hara Thomas J , Phartiyal Pallavi , Wang Jinling , He Jia-Qiang , Rudy Yoram , Ackerman Michael J
Published: July 2021

Cardiac I
Kr
is a critical repolarizing current in the heart and a target for inherited and acquired long QT syndrome. Biochemical studies show that native I
Kr
channels are heteromers composed of both hERG 1a and 1b subunits, yet our current understanding of I
Kr
functional properties derives primarily from studies of homo-oligomers of the original hERG 1a isolate. The hERG 1a and 1b subunits are identical except at the amino (NH2) terminus, which in hERG 1b is much shorter and has a unique primary sequence. We compared the biophysical properties of currents produced by hERG 1a and 1a/1b channels expressed in HEK-293 cells at near-physiological temperatures. We found that heteromeric hERG 1a/1b currents are much larger than hERG 1a currents and conduct 80% more charge during an action potential. This surprising difference corresponds to a two-fold increase in the apparent rates of activation and recovery from inactivation, which reduces rectification and facilitates current rebound during repolarization. Kinetic modeling shows these gating differences account quantitatively for the differences in current amplitude between the two channel types. Depending on the action potential model used, loss of 1b predicts an increase in action potential duration of 27 ms (7%) or 41 ms (17%), respectively. Drug sensitivity was also different. Compared to homomeric 1a channels, heteromeric 1a/1b channels were inhibited by E-4031 with a slower time course and a corresponding four-fold positive shift in the IC
50
. Differences in current kinetics and drug sensitivity were modeled by “NH2 mode” gating with conformational states bound by the amino terminus in hERG 1a homomers but not 1a/1b heteromers. The importance of hERG 1b
in vivo
is supported by the identification of a 1b-specific A8V missense mutation in 1/269 unrelated genotype-negative LQTS patients and absent in 400 control alleles. Mutant 1bA8V expressed alone or with hERG 1a in HEK-293 cells nearly eliminated 1b protein. Thus, mutations specifically disrupting hERG 1b function are expected to reduce cardiac I
Kr
, prolong QT interval and enhance drug sensitivity, thus representing a potential mechanism underlying inherited or acquired LQTS.
Abstract 5317: Identification and Characterization of a Novel Susceptibility Gene,
SCN3B,
for Idiopathic Ventricular Fibrillation
Authors: Valdivia Carmen R , Mereidos-Domingo Argelia , Algiers Thimothy J , Ackerman Michael J , Makielski Jonathan C
Published: July 2021

Background:
Mutations in the Na
V
1.5 sodium channel macromolecular complex have been identified in some cases classified as idiopathic ventricular fibrillation (IVF). IVF and Brugada syndrome (BrS) are partially overlapping syndromes. Here, we report a mutation in
SCN3B-
encoded sodium channel β3 subunit as a novel pathogenic mechanism for IVF.

Methods:
Comprehensive open reading frame mutational analysis of
SCN5A, GPD1L,
and the beta subunit genes (
SCN1–4B
) was performed using PCR, DHPLC, and direct DNA sequencing of DNA extracted from a 20-year-old patient diagnosed with IVF. The
SCN3B
mutation was made by site directed mutagenesis and co-transfected with SCN5A into HEK-293 cells for functional chraracterization using the patch clamp technique.

Results:
A novel missense mutation, V54G-SCN3B, was identified in a 20-year-old male following collapse and external defibrillation from VF. After recovery, there was no detectable electrocardiographic abnormality. Imaging studies demonstrated a structurally normal heart, and the patient was diagnosed with IVF. The mutation was absent in 800 reference alleles and involved a highly conserved residue in the extracellular domain of the beta 3 subunit. No other mutations were identified in the 5 other genes. HEK cells expressing SCN5A and either WT-, or V54G-SCN3B were studied 24 hours after transfection. Cells expressing V54G-SCN3B showed significant decrease in sodium current density of 60±20 pA/pF compared to 203±35 pA/pF in WT-SCN3B (n=14–19). In addition V54G-SCN3B significantly shifted the activation curve +5 mV without affecting inactivation.

Conclusions:
This study provides the first molecular and cellular evidence implicating
SCN3B
in IVF. Given the marked loss-of-function to the sodium channel by V54G-SCN3B and the overlap between IVF and BrS, it will be interesting to determine whether mutations in
SCN3B
explain some cases of genotype negative Brugada syndrome.
Abstract 5326: S100A1 Prevents Arrhythmogenic Diastolic Sarcoplasmic Reticulum Calcium Leak In Ventricular Cardiomyocytes
Authors: Voelkers Mirko , Weidenhammer Christian , Herzog Nicole , Friedrich Oliver , Fink Rainer H , Koch Walter J , Katus Hugo A , Soltys Steven S , Rabinowitz Joseph , Most Patrick
Published: July 2021

Background:
S100A1 is an inotropic calcium (Ca) sensor protein in cardiomyocytes interacting with the cardiac ryanodine receptor (RyR2). Previous studies of our group have shown that S100A1 protein can decrease Ca spark frequency in permeabilized ventricular cardiomyocytes (CMs). We therefore hypothesized that S100A1 might prevent arrhythmogenic sarcoplasmic reticulum (SR) Ca leak in CMs.

Methods and Results:
Ventricular rat cardiomyocytes were enzymatically isolated and transfected either with 10 MOI of a CMV-GFP control or CMV-S100A1/CMV-GFP adenovirus resulting in GFP fluorescence in >99% of cultured GFP-CMs and S100A1-CMs as assessed by epifluorescent microscopy after 24-hours. Expression analysis revealed a 3-fold increase in S100A1 protein in S100A1-CM levels compared to GFP-CM. Confocal line-scan microscopy yielded a 50% reduction in Ca spark frequency in rhodamin2-AM loaded quiescent S100A1-CMs and epifluorescent imaging showed a 20% and 50% increase in the SR Ca content and systolic Ca transient amplitude, respectively, in FURA2-AM loaded 2Hz-stimulated S100A1-CM (37°C, 1.8 mM Ca) compared with GFP-CMs. Pathophysiological diastolic SR Ca leak was induced subjecting 2Hz-stimulated FURA2-AM loaded CMs to 0.5 mM caffeine and 10–7 M isoproterenol (caff/iso) as previously published by Isner and co-workers. As expected, this protocol resulted in permanent diastolic Ca waves in 100% (n=60/60) of 2Hz-stimulated iso/caff-treated GFP-CMs and was accompanied by significantly increased diastolic Ca levels and reduced systolic Ca transient amplitudes compared with iso-treated GFP-CMs. However, S100A1 efficiently protected more than 80% of iso/caff treated S100A1-CMs (n=49/60 cells, P<0.05 vs. iso/caff GFP-CMs) from diastolic Ca waves and further alterations of CM Ca handling. Importantly, similar results were obtained in caff/iso-treated failing rat GFP- and S100A1-CMs (data not shown).

Conclusions:
Here we show for the first time that S100A1 can both diminish the physiological SR Ca leak and protect from arrhythmogenic Ca waves in CMs. Given its beneficial effects in the context of experimental HF animal models, S100A1 therapeutic inotropic actions might be complemented by an antiarrhythmic effect targeting dysfunctional RyR2.