bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3726: Right Ventricular Microcirculatory Changes in Pulmonary Hypertension-Associated Right Heart Failure Imaged by Three-Dimensional Confocal Microscopy
Authors: Bogaard Harm J , Henderson Scott C , Natarajan Ramesh , Kraskauskas Donatas , Voelkel Norbert F
Published: July 2021

Background:

Right ventricular (RV) ischemia occurs in pulmonary hypertension (PH) patients despite normal coronary angiograms. O
2
supply-demand balance in the pressure-overloaded RV is determined by the interplay between reduced coronary perfusion (due to systemic hypotension and compression of coronary vessels) and a wall stress-related increase in myocardial O
2
consumption. The role of capillary rarefaction, which is involved in pressure overload induced
left
heart failure, has not been investigated in PH-associated RV failure.

Methods:

Rats received a single dose of the VEGF receptor blocker SU5416 and were exposed to 4 wks of normobaric hypoxia and 2 wks of sea level room air. This regimen induces angioproliferative changes in the pulmonary circulation, severe PH and RV failure. At 6 wks, the rats received intravital injections of Texas-Red conjugated tomato lectin, which is an N-acetylglucosamine specific marker of blood vessels in rodents. After circulation of the lectin for 5 min, papaverin was injected to promote maximal vasodilation of all perfused blood vessels. Animals were exsanguinated and perfusion-fixed with paraformaldehyde. After overnight fixation, 300 μm thick transverse sections of the myocardium were bleached in a methanol/DMSO mixture and nuclei were stained using DAPI. Prior to 3-dimensional imaging under a confocal microscope, further tissue clearing was achieved using organic solvents.

Results:

Z-stacks of 2-dimensional images were made in whole-mount cardiac specimens, using a step size of 0.32 μm. 3-dimensional reconstruction allowed quantification of the total capillary volume, which was reduced in the PH model compared to controls (2.9±1% vs. 5.7±1% of tissue volume, p<0.05). The reconstructions showed a remarkable increased morphological heterogeneity of the capillaries in the failing RV. In contrast, images of the LV microcirculation in the SU5416 treated animals were not different from those of controls.

Conclusions:

RV failure in experimental PH is associated with capillary rarefaction. 3-dimensional imaging of the myocardial microcirculation can be achieved using intravital injections of tomato lectin and subsequent confocal imaging.
Abstract 3744: Local Transplantation Of Blood-derived Late-outgrowth Progenitor Cells Increased Reendothelialization, Inhibited Smooth Muscle Cell Proliferation And Reduced Neointima Formation After Experimental Carotid Injury
Authors: Schurmann Maren , Knoedler Martina , Fischer Philipp , Peschel Christian , Oostendorp Robert , Ott Ilka
Published: July 2021

Introduction
: Recent studies demonstrate the vasculoprotective effects of cell-based therapies to inhibit limit restenosis. Delivery of autologous late outgrowth endothelial progenitor cells (eEPC) result in early reendothelialization and inhibition of neointimal hyperplasia. Additional anti-inflammatory treatment may further reduce neointima generation.

Methods and Results
: CD34+ cells were isolated using immunomagnetic beads and cultured in endothelial cell medium to obtain eEPC. Early passage cells were transduced ex vivo by a retroviral vector expression of IL-1ra (eEPC IL-1ra) or empty vector (eEPC pLXSN) and expanded up to 46 population doublings. Expression levels were confirmed by RT-PCR. Athymic nude rats underwent carotid balloon and wire injury immediately followed by local delivery of eEPCs, eEPC IL-1ra, eEPC pLXSN or buffer for 20 minutes (n=22). Elastica van Giesson staining revealed a decrease in the intima/media ratio from 1.53 ± 0.11 to 1.13 ± 0.09 (mean ± SEM) after transplantation of eEPC (p=0.009). However, no additional reduction in neointima generation was observed after transplantation of eEPC IL-1ra. Endothelial-cell specific staining demonstrated an enhancement of reendothelialisation by eEPCs 24 hours after vascular injury. After 2 weeks a reduction of Ki67+ proliferating cells from 37.0 ± 8.5 Ki67+ cells in the control group to 11.67 ± 2.85 Ki67+ cells after eEPC transplantation (P=0.03) was found. Yet, no additional anti-proliferative effects were observed after transplantation of eEPC IL-1ra. Thus, enhancement of reendothelialization through local delivery of eEPCs may be sufficient for the inhibition of neointima generation since additional anti-inflammatory treatment using eEPC IL-1ra had no beneficial effect.

Conclusion
: Local delivery of blood-derived expanded endothelial progenitor cells after vascular injury contributes to endothelial regeneration, inhibits neointimal smooth muscle cell proliferation and reduces neointima generation. Autologous cell transplantation after vascular injury may be a feasible strategy for promotion of reendotheli-alisation and improvement of vascular regeneration.
Abstract 3759: Enhanced External Counterpulsation Arrests the Progression of Atherosclerosis by Regulating Cellular Apoptosis and Proliferation Signaling Pathway
Authors: Zhang Yan , Ma Hong , He Xiaohong , Wu Guifu , Chen Xiaolin , Liu Donghong , Du Zhimin , Dong Yugan , Jin Yafei , He Wen , Wang Kuijian , Luo Jinyun , Xiong Yan , Lawson William E , Hui John C , Zheng Zhensheng
Published: July 2021

Shear stress may be the most crucial local factor affecting atherogenesis. Enhanced External Counterpulsation (EECP) has been documented to increase arterial wall shear stress in vivo. We hypothesize that chronic exposure to increased pulsatile shear stress by EECP arrests the progression of atherosclerosis due to hypercholesterolemia. Forty-eight pigs were randomized to usual diet (n = 7), high-cholesterol diet (n = 18), and high-cholesterol diet plus EECP (34±2 hours, n = 23). At week 15, the left anterior descending coronary arteries (LAD) were collected for HE staining, transmission electron microscopy examination, immunohistochemical analysis of AKT, caspase- 3 and TUNEL assay. Expression and localization of NK- κB was assessed by immunofluorescence and confocal laser scanning microscope. The aortas were isolated for Sudan staining and Western-blot analysis. Blood lipids, viscosity, Doppler ultrasound flow examination was performed. Statistical analysis was by Kruskal-Wallis and Mann-Whitney tests. During EECP, the peak diastolic arterial wall shear stress in the right brachial artery increased significantly compared with baseline (53.18±3.81 dynes/cm
2
versus 23.92±2.02 dynes/cm
2
, p<0.001), as well as the mean arterial wall shear stress (27.46±2.22 dynes/cm
2
versus 20.43±1.87 dynes/cm
2
,
p
< 0.001). High-cholesterol diet induced remarkable atherosclerotic lesions with excessive cellular proliferation and apoptosis. In comparison, hypercholesterolemic animals receiving EECP showed ameliorated atherosclerotic lesions in LADs ( lesion/medial area ratio: 19.71±3.79 % versus 7.32±1.59 %,
p
= 0.001) and aortas (percentage of plaque area: 9.95±1.81 % versus 5.16±1.08 %,
p
= 0.011), coupled with decreased TUNEL positive staining (64±2.08 % versus 49±2.27 %,
p
< 0.001), reduced caspase- 3 protein expression, AKT and NF- κB activity. This study demonstrates that EECP administration leads to reduced atherosclerotic lesions, coupled with dual inhibitory effects on vascular cellular proliferation and apoptosis. Its mechanisms are related to chronic increased shear stress by EECP suppressing the over-activation of the AKT/NF-κB signaling pathway.
Abstract 3760: Atherogenic Affects Of Arsenic: Role Of Endoplasmic Reticulum Stress And Unfolded Protein Response
Authors: Haberzettl Petra , Vladykovskaya Elena , Barski Oleg , Sithu Srinivas , D’Souza Stanley , States Christopher , Srivastava Sanjay
Published: July 2021

Arsenic is a global water contaminant and EPA has listed arsenic as a high priority hazardous substance in the United States. Epidemiological studies suggest that chronic arsenic ingestion increases cardiovascular disease in humans, particularly, carotid atherosclerosis. However, mechanisms of arsenic-induced atherogenesis are unknown. We examined the effect of arsenic exposure on early lesion formation in apoE-null mice maintained on water supplemented with (0, 1, 5 and 50 ppm; 3–16 weeks of age) sodium arsenite. Arsenic, did not affect plasma cholesterol but decreased the triglycerides by 18±4 % (P<0.05). NMR analysis of the lipoproteins showed a significant decrease in the abundance of large VLDL particle (>60 nm diameter). Despite a significant decrease in plasma triglyceride, atherosclerotic lesion formation was significantly increased (2– 4 fold; P<0.05 for all doses) in the aortic sinus and the aortic arch of the arsenic-fed mice in a dose dependent manner. Immunohistochemical analysis showed significant increase in the accumulation of macrophages, expression of MCP-1 and unfolded protein response (UPR) dependent activating transcription factor (ATF)-4 and ATF3, in the lesions of arsenic (1ppm) exposed mice.
In vitro
, arsenic (5–25 μM), significantly increased the expression of ICAM-1, transmigration of differentiated monocytes and expression of the pro-inflammatory cytokine IL-8 in vascular endothelial cells (vEC). Arsenic, also increased the expression of ER-chaperones Grp 78, HERP and calnexin (2– 6 fold; P<0.01). Examination of the effect of arsenic on UPR showed that arsenic, induced the splicing of IRE-1 dependent, bZIP transcription factor XBP-1(alarm phase) and increased the phosphorylation of eIF2α (PERK mediated adaptive phase) by 3 fold (P<0.01) in vEC. Arsenic also induced the expression of the downstream effecter proteins of eIF2α-ATF3 (8 fold; P<0.01) and pro-apoptotic protein CHOP (4 fold; P<0.01) in vEC. Chemical chaperone, phenyl butyric acid (PBA), attenuated the arsenic-induced expression of ATF3 (>90%; P<0.001) and CHOP (>90%; P<0.001). These data suggest that ER-stress and UPR could exacerbate arsenic-induced vascular inflammation and promote atherogenesis.
Abstract 3770: Concentration-dependent Divergent Hypertrophic Effects of Estrogen in Adult Ventricular Myocytes
Authors: Kilic Ana , Javadov Sabzali A , Karmazyn Morris
Published: July 2021

High concentrations of estrogen have been shown to attenuate myocardial hypertrophy and left ventricular remodeling. However, the effects of low concentration of estrogen observed in postmenopausal women on cardiac hypertrophy have not been studied. In the present study we examined the effects of high (0.1 and 1 nM) and low (1 and 10 pM) concentration of the synthetic analog of estradiol, 17β-estradiol (E2) on adult cardiomyocytes (CMs). CMs were isolated from adult male and female Sprague-Dawley rats. The cells were used immediately after isolation to measure pH
i
or cultured to assess hypertrophic phenotype (cell surface area), gene markers (atrial natriuretic peptide, ANP), and protein activation. Low concentration of E2 (1 and 10 pM) increased cell surface area (females: 20%,
P
<0.05; males: 28%,
P
<0.05) and ANP expression (females: 394%,
P
<0.05; males: 497%,
P
<0.05) after 24 h. However, high concentrations (0.1 and 1 nM) of E2 did not induce cell hypertrophy but instead blocked the hypertrophic effect of the α
1
-agonist phenylephrinerophy. The pro-hypertrophic effect of low concentration of E2 was prevented by the sodium-hydrogen exchange isoform 1 (NHE-1) specific inhibitor AVE-4890 (AVE, 5 μM) suggesting involvement of NHE-1 in mediating the E2-induced hypertrophy. Fluorometric measurements with the pH
i
-sensitive dye BCECF demonstrated that a 1 pM E2 increased the pH
i
(females: +0.05 pH units; males +0.12 pH units,
P
<0.05) by a rapid non-genomic mechanism that was blocked by AVE. On the other hand, 1 nM E2 decreased the pH
i
(females: −0.24 pH units,
P
<0.05; males: −0.07 pH units,
P
<0.05) and this effect was also prevented by AVE. The NHE-1-mediated pro-hypertrophic effect of 1 pM E2 was dependent on phosphorylation of ERK1/2 MAPK since the effect was blocked with the ERK1/2 inhibitor PD98059 (10 μM) and there was no gender difference on ERK1/2 activation. E2 has a dual concentration-dependent role in adult CMs as manifested by a pro-hypertrophic effect at low concentrations (1 and 10 pM), and conversely, an anti-hypertrophic effect at high concentrations (0.1 and 1 nM). The pro-hypertrophic effect of E2 is mediated, at least in part, through ERK1/2/NHE-1 activation.
Abstract 3774: A Novel Protein Phosphatase in SR Calcium and Cardiac Contractile Regulation
Authors: Ruan Hongmei , Lu Gang , Wang Yibin
Published: July 2021

Loss of contractility is a common feature in heart failure. Sarcoplasmic Reticulum (SR) calcium cycling defect is a major mechanism underlying contractile dysfunction. Phospholamban (PLB) mediated regulation of SERCA activity in normal and failing heart plays an important role in SR calcium cycling. Recently, we discovered a novel isoform of protein phosphatase 2C family (PP2Ce) which is specifically targeted on ER membrane. Northern-blot showed that PP2Ce is highly expressed in heart and other tissue. Expression of PP2Ce in neonatal cardiomyocytes significantly repressed the PLB phosphorylation at the Thr-17 site without remarkable effect on Ser-16 site, which indicating its potent and very specific activity towards the Thr-17 site instead of the Ser-16 site of PLB in cardiomyocytes. By using an inducible a-MHC-in-PP2Ce construct and the Tet-expresser transgenic line, we developed a cardiac specific PP2Ce transgenic mice. PP2Ce expression was remarkably higher in transgenic hearts compared with control, accompanying with the increase in expression of phosphor-CaMKII. There were no significant difference in the cardiac function at basal based on Echocardiography measurements. Expression of PP2Ce markedly delayed the decay of calcium transients with the administration isoproterenol in isolated adult cardiomyocytes and blunted positive inotropic response in transgenic mice heart. In response to ISO treatment, PLB phosphorylation at the Thr-17 site were significantly suppressed in PP2Ce cardaiomyocytes and hearts, while there is no much change at Ser-16 site compared with controls. Our results suggested that PP2Ce may be a novel component of the PLB/SR calcium regulatory machinery involved in cardiac contractile regulation under physiological and pathological conditions and offer new insights to cardiac contractile regulation and failure.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 3787: Effects of Running Exercise on Aortic Telomere Regulating Proteins, Survival Pathways and Apoptosis
Authors: Werner Christian , Hanhoun Milad , Poess Janine , Fuerster Tobias , Haendeler Judith , Boehm Michael , Laufs Ulrich
Published: July 2021

Background:
The vasoprotective effects of physical exercise are only partially understood on the molecular level. Telomere-associated proteins affect cellular senescence and survival. We studied the molecular effects of physical exercise in aortic tissue of running and sedentary mice.

Methods:
C57/Bl6 wildtype, endothelial nitric oxide synthase (ENOS) deficient and telomerase reverse transcriptase (TERT) deficient mice were randomized to voluntary running or no running wheel conditions (n=8 –10 per group).

Results:
Physical exercise for 3 weeks upregulated aortic telomerase activity to 303 ± 32% (p<0.01) of sedentary controls, increased protein expression telomere-repeat binding factor (TRF) 2 (254 ± 35%, p<0.05) and reduced protein expression of the senescence / apoptosis regulators Chk2 (41 ± 12%), p53 (51 ± 12%) and p16 (64 ± 19%). Running increased mRNA expression of TRF1 (193 ± 50%), TRF2 (192 ± 14%) and of the repair protein Ku70 (365 ± 121%). These exercise-induced changes were absent in ENOS −/− and TERT −/− mice. Aortic telomere length as determined by quantitative fluorescence in-situ hybridization (QFISH) did not differ between 3 week and 6 months old sedentary or running mice (1914 ± 166 vs. 1762 ± 394 telomere fluorescence units, TFU), but was lower in 18 months old sedentary control mice (1142 ± 117 TFU, p<0.05). To test the physiologic relevance of these exercise-mediated anti-senescent pathways, vascular oxidative stress was induced in-vivo by intraperitoneal injection of 25mg/kg paraquat for 24 hours before quantification of endothelial apoptosis in the thoracic aorta. Compared to sedentary mice, paraquat increased apoptotic cell death to 581 ± 48 %, whereas apoptosis was significantly lower in mice preconditioned by 21 days voluntary exercise (320 ± 34%, p<0.001 vs. control and paraquat).

Conclusions:
Voluntary physical activity upregulates aortic telomere regulating proteins which induces anti-senescent and anti-apoptotic effects mediated by ENOS and TERT independent of telomere length.
Abstract 3809: Cardiac-Specific Overexpression of EcSOD Using an AAV9 Vector in Combination with the Cardiac Troponin-T Promoter Inhibits LV Remodeling after Myocardial Infarction
Authors: Prasad Konkal-Matt R , Beyers Ronald J , Xu Yaqin , French Brent A
Published: July 2021

Introduction:
The wide tissue tropism exhibited by AAV provides for efficient gene transfer throughout the body, but targeting gene expression to cardiomyocytes is desirable for cardiac gene therapy. We hypothesized that targeted overexpression of extracellular superoxide dismutase (EcSOD) via the cardiac Troponin-T (cTnT) promoter would suffice to minimize left ventricular (LV) remodeling after myocardial infarction (MI).

Methods:
An AAV9 vector expressing EcSOD from the cTnT promoter (AcTnTEcSOD) was injected into 5 wk-old C57 mice via jugular vein (3x10
11
vp/mouse). Western blots, immunohistochemistry &
in vitro
SOD assays were used to measure EcSOD expression, distribution and activity. Cardiac magnetic resonance (CMR) imaging was performed at baseline (5 wks post-vector injection) and at days 1, 7 & 28 after MI to assess LV volumes (vol) & ejection fraction (EF) as compared to WT mice (n=4). Infarct (IF) sizes were also compared by DE on D1.

Results:
Systemic injection of the vector (AcTnTEcSOD) provided uniform EcSOD overexpression within cardiomyocytes (Panels A&B) and elevated total cardiac SOD activity by 5.6 fold (p<0.05). On D1 post-MI, IF sizes were similar in vector & WT groups (p=ns). The vector group had significantly lower end-diastolic vol at D7, D28 and lower end-systolic vol at D28 (all p<0.05 by ANOVA, Panels C&D), resulting in improved D28 EF over controls (p=0.02).

Conclusions:
Cardiac-specific overexpression of therapeutic genes can be achieved by combining highly-efficient AAV9 vectors with cardiac-specific promoters. AAV-mediated, cardiac-restricted overexpression of EcSOD from the cTnT promoter significantly reduces post-MI LV remodeling.
Abstract 3815: Pim-1 Gene Delivery to Cardiac Progenitor Cells Prevents Long Term Cardiac Failure
Authors: Fischer Kimberlee , Wu Weitao , Cottage Christopher , Gude Natalie , Sussman Mark
Published: July 2021

Cardiac stem cell therapy administered at the time of infarction effectively blunts cardiomyopathic damage a few weeks after treatment but long term efficacy of adoptive stem cell transfer remains largely unknown. A comparative analysis shows that long term (25 week) protection is only provided by Pim-1 modified progenitor cells whereas unmodified progenitor cells fail to provide prolonged benefits after six weeks post intramyocardial injection. After infarction mice were intramyocardially injected with saline, unmodified cardiac progenitor cells (CPC’s), or Pim-1 modified CPC’s. At a 12 week time point mice injected with Pim-1 modified CPC’s have increased EF and FS as compared to mice injected with unmodified cells (n=15 for each group). Invasive hemodynamic measurements (n=5 for each group) confirm that mice injected with Pim-1 modified CPC’s had improved performance by assessment of multiple functional criteria compared to unmodified cells. Longitudinal monitoring by echocardiography reveals that mice injected with Pim-1 modified CPC’s sustain fractional shortening and ejection fraction for up to 24 weeks whereas animals that receive unmodified cells begin to fail within 6 weeks after delivery and were not statistically different than saline injected controls after 12 weeks. In addition Pim-1 CPC injected mice had infarct sizes 52% smaller than control groups (p<.02). Both saline and unmodified CPC’s groups maintained similar infarct sizes (~60% of left ventricular free wall). Confocal microscopy reveals engraftment and persistence of injected Pim-1 CPC’s into the myocardium with differentiation into multiple cardiogenic lineages giving rise to myocytes, vasculature, and endothelium Successful long term functional improvement of the heart requires the engraftment of Pim-1 genetically modified cardiac progenitor cells. Unmodified cells offer an acute ameliorative response but ultimately fail in the ability to effectively repair and maintain the function of the heart. These results support use of genetic engineering to enhance stem cell-mediated myocardial regeneration.
Abstract 3816: Injection of GSK-3β Overexpressing Bone Marrow Derived Mesenchymal Stem Cells Attenuates Cardiac Dysfunction after Myocardial Infarction
Authors: Cho Jaeyeaon , Zhai Peiyong , Gao Shumin , Sadoshima Junichi
Published: July 2021

Glycogen synthase kinase 3β (GSK-3β) induces expression of cardiac markers in mesenchymal stem cells (MSCs). We examined whether injection of
ex vivo
-modified GSK-3β overexpressing MSCs improves cardiac dysfunction after myocardial infarction (MI). Mouse MSCs were transduced with adenovirus harboring LacZ or GSK-3β (LacZ-MSC or GSK-MSC). MI was induced by coronary ligation and thirty μL of saline alone or MSCs (1.5 x 10
5
cells) were injected into the MI border zone just after coronary ligation. In saline injected MI mice, a progressive increase in left ventricular end diastolic dimension (LVEDD) and a progressive decrease in LV ejection fraction (LVEF) and LV fractional shortening (%FS) were observed compared with sham (Table

). LacZ-MSC or GSK-MSC attenuated increases in LVEDD at 2 and 12 weeks. However, LVEF and %FS were significantly greater in GSK-MSC than in LacZ-MSC group at 2 and 12 weeks (Table

). Heart weight/body weight (HW/BW) in GSK-MSC (4.4±0.1) group was significantly smaller than that in LacZ-MSC (5.1±0.2) and saline (5.0±0.2) groups and not significantly different from that in sham group (4.1±0.0), suggesting that cardiac remodeling was attenuated by GSK-MSC. To further confirm the effect of GSK-3β, MSCs acquired from inducible GSK-3β (tet-OFF) mice were injected into the MI mice fed with or without doxycycline (Dox), where expression of GSK-3β transgene was abrogated by Dox treatment
in situ
. After two weeks of MI, both LVEF (61.5±2.2, 37.4±7.1, p<0.01) and %FS (27.6±1.4, 15.4±3.2, p<0.001) were significantly greater in Dox (−) (n=13) than in Dox (+) mice (n=9). These results suggest that
ex vivo
introduction of GSK-3β improves the effect of MSC injection, thereby attenuating LV dysfunction after MI. Furthermore,
in situ
Dox-regulatable expression of GSK-3β also facilitated the therapeutic effect of MSCs injection.

Echocardiography on 2 Weeks and 12 Weeks
Abstract 3821: Mesp1 Mediates Cardiovascular Differentiation By Dkk-1 Driven Inhibition Of Wnt-signaling And Is A Suitable Tool For Isolation Of Es Cell Derived Cardiovascular Progenitors
Authors: David Robert
Published: July 2021

We have recently shown that MesP1 is a cardiac key transcription factor sufficient to induce ectopic beating heart tissue. MesP1 overexpression in ES cells results in significantly increased cardiovasculogenesis. Our results revealed a prominent function of MesP1 within a gene regulatory cascade causing Dkk-1 mediated blockage of wnt signalling. MesP1 may become a tool to preprogram ES cells for cardiac cell therapy and tissue engineering. In addition to directed differentiation, future cardiac cell therapy strategies will require protocols for a high-yield isolation of specific cell types to avoid the hazard of teratoma and tumor formation. Therefore we have developed a method for magnetic cell sorting (MACS) of ES cells expressing a human CD4 surface marker lacking its intracellular domain (ΔCD4) under control of transgenic promoters. Here we show that the MACS system can successfully be transferred to a MesP1 promoter-ΔCD4 construct. After transfection into murine ES cells several stable clones were obtained in which the time course of ΔCD4-expression revealed a peak at days two and three of differentiation. Using MACS we were able to enrich this population to purities of over 97%. The MACS-positive fractions showed a strong enrichment of the endogenous murine MesP1 mRNA confirming the functionality of our construct. In reaggregation experiments after MACS we verified the viability of the sorted cells which could be further propagated in their primitive differentiation state in the presence of LIF. After LIF withdrawal subsequent spontaneous beating activity was approximately 10 fold increased. This effect is reflected by an upregulation of cardiovascular mRNAs and on the protein level (FACS) by a 3 to 10 fold increased appearance of TroponinI, α-MHC and CD31 expressing cells. The cardiomyocytes revealed normal expression patterns of sarcomeric proteins. Whole Cell Patch Clamp analyses showed action potentials characteristic for early type cardiomyocytes corresponding to embryonic hearts at ED 10. These results suggest that cardiovascular progenitor cells can be selected from ES cells via MACS purification based on MesP1 promoter driven ΔCD4 expression. These cells are predestined for cell transplantation and tissue engineering approaches.
Abstract 3858: Micropatterned Ventricular Slice: Role of Realistic Tissue Microstructure in Impulse Conduction
Authors: Badie Nima , Bursac Nenad
Published: July 2021

We developed novel cultures of neonatal rat cardiomyocytes that accurately replicate the MRI-measured microstructure of murine ventricular cross-sections. The individual roles of realistic tissue boundaries and changes in local myofiber direction in impulse conduction were studied by optically mapping membrane voltage (Fig A1

) in (1) micropatterned cultures with realistic tissue boundaries and natural (anisotropic) cell orientation (Fig A2

) and (2) controls with the same boundaries but random (isotropic) cell orientation (Fig A3

). Compared to isotropic controls, anisotropic cultures exhibited a 27% larger spatial dispersion of conduction velocity (CV) at 2 Hz pacing, 17% faster rise in CV dispersion during ramp pacing (5–14 Hz), and 100% higher incidence of remote conduction block, which occurred exclusively at the junctions between the septum and right ventricle (RV) free wall due to current source-sink mismatches and sharp turns in cardiac fiber directions. The incidence of block with ramp pacing was pacing site dependent from the RV, septum, and left ventricle (LV), yielding remote blocks in 77%, 38%, and 0% of anisotropic and 33%, 25%, and 0% of isotropic cultures (Fig B

). In anisotropic cultures, the cycle length of resulting anatomical reentries (normalized by CV) was 34% shorter in LV- vs RV-driven reentry (Fig C1–2

), due to a shorter wave path. Overall, realistic tissue boundaries and fiber directions synergistically increase the non-uniformity of conduction, render specific regions highly susceptible to conduction block, and determine reentry rate. These purely structural effects are independent of the presence of regional variations of cell ionic properties.

This research has received full or partial funding support from the American Heart Association, AHA Mid-Atlantic Affiliate (Maryland, North Carolina, South Carolina, Virginia & Washington, DC).
Abstract 3894: Inhibition of iNOS Uncoupling by Tetrahydrobiopterin Confers Cardioprotection through Protein
S-
Nitrosylation in Streptozotocin-Induced Diabetic Rat
Authors: Okazaki Toru , Otani Hajime , Yamashita Koji , Jo Hiromi , Yoshioka Kei , Moriguchi Akira , Iwasaka Toshiji
Published: July 2021

Although expression of inducible nitric oxide synthase (iNOS) and oxidative stress are increased in diabetic (DM) hearts, the role of iNOS uncoupling in ischemia/reperfusion (IR) injury remains unknown. Because iNOS-derived NO is known to play a crucial role in cardioprotection against IR injury in non-DM hearts, we hypothesized that iNOS uncoupling may compromise tolerance to IR injury in the DM heart by decreasing the bioavailability of NO. The expression and activity of iNOS but not n/eNOS were increased in the streptozotocin-induced DM rat heart. Under Langendorff perfusion, superoxide generation as evaluated by dihydroethidium accumulation in the nucleus was significantly increased in cardiomyocytes of the DM heart, but it was inhibited by treatment with the NOS co-factor tetrahydrobiopterin (BH4; 10 μM) or an iNOS selective inhibitor 1400W (10 μM). BH4 increased NOx, a marker of NO bioavailability, and cGMP in the DM heart. The increase in cGMP by BH4 was abrogated by co-treatment with 1400W or a NO-sensitive guanylyl cyclase inhibitor ODQ (10 μM). BH4 significantly decreased nitrotyrosin formation but increased protein
S
-nitrosylation in the DM heart. The increase in protein
S
-nitrosylation by BH4 was abolished by co-treatment with a thiol reducing agent dithiothreitol (DTT; 5 mM). The isolated rat heart was subjected to 30 min global ischemia followed by 120 min reperfusion. Post-ischemic recovery of left ventricular (LV) function and infarct size was comparable between the non-DM and the DM hearts. Pre-ischemic treatment with BH4 significantly improved post-ischemic LV function and reduced infarct size only in the DM heart. Co-treatment with BH4 and 1400W, ODQ, or DTT had no significant effect on post-ischemic LV function and infarct size in the non-DM heart. However, co-treatment with BH4 and 1400W or DTT but not ODQ abolished BH4-induced improvement of post-ischemic LV function and reduction of infarct size in the DM heart. These results suggest that inhibition of iNOS uncoupling by BH4 confers cardioprotection against IR injury in the streptozotocin-induced DM rat heart by increasing the bioavailability of NO and this cardioprotective effect is mediated by protein
S
-nitrosylation but not cGMP.
Abstract 3899: SDF-1α Mediates the Therapeutic Effect of Human Adipose-Derived Stem Cells on Acute Myocardial Infarction Recruiting Bone Marrow-Derived Endothelial Progenitor Cells
Authors: Ii Masaaki , Yokoyama Ayumi , Horii Miki , Akimaru Hiroshi , Asahara Takayuki
Published: July 2021

Background:
Recently, human multipotent adipose-derived stem cells (hMADSs) have been isolated featuring extensive expansion capacity ex vivo. However, little is known about the therapeutic efficacy of hMADS in ischemic heart diseases. We tested the hypothesis that hMADS transplantation may contribute to cardiac functional recovery following myocardial infarction (MI).

Methods and Results:
Nude rats were either transplanted with hMADSs (5x10
5
/rat, n=10) or PBS (control, n=9) in ischemic myocardium immediately following MI induction. The cardiac function, infarct size and capillary density in the peri-infarct area were evaluated by echocardiography and immunostaining 28 days after surgery. The cardiac function was significantly greater with increased capillary density and reduced fibrosis area in the hMADS group than that in the control group. Next, we examined tissue regeneration in the infarct heart by the transplanted hMADSs. However, remarkable differentiation of hMADSs into any cardiac cell lineages was not detected. To explore another mechanism for the favorable effect of hMADSs, we further examined mRNA expression of cytokines in hMADSs under hypoxic conditions. Although hypoxia decreased the expressions, robust VEGF, bFGF, and SDF-1α expressions were detected in hMADSs. Notably, the stem/progenitor chemokine SDF-1α expression in hMADSs was significantly greater than that in human mesenchymal stem cells that are well known to have a therapeutic effect on ischemic heart diseases. We then focused on SDF-1α /CXCR4 axis and examined the contribution of bone marrow (BM)-derived endothelial progenitor cells (EPCs), that have CXCR4 receptor for SDF-1v, to ischemic myocardium using a Tie2/LacZ BM transplantation nude mouse model. β-gal positive EPCs are frequently observed in ischemic myocardium in the hMADS group compared to the control group.

Conclusion:
hMADSs exhibit a therapeutic effect on cardiac function following MI with the production of VEGF, bFGF, and SDF-1α demonstrating paracrine effects rather than direct contribution to cardiac regeneration. These findings suggest that transplanted hMADSs and recruited EPCs may synergistically promote angiogenesis playing a role in ischemic myocardium.
Abstract 3917: Role of Endothelial Cell-Selective Adhesion Molecule in the Progression of Atherosclerosis
Authors: Inoue Michihiko , Ishida Tatsuro , Hara Tetsuya , Husni Cangara M , Sun Li , Hirata Ken-ichi
Published: July 2021

Backgrounds
: Endothelial cell-selective adhesion molecule (ESAM) is a new member of the immunoglobulin superfamily, which is expressed in vascular endothelial cells. Although ESAM has been shown to mediate homohilic adhesion between endothelial cells, the interaction of ESAM and hematopoietic cells has not been ingestigated. Also, the role of ESAM in atherosclerosis remains unclear. In this study, we assessed the role of ESAM in monocyte/ macarophage infiltration, and examined effects of ESAM inactivation in the development atherosclerosis using a murine model of atherosclerosis.

Methods and Results
: ESAM−/− mice were bred with apoE−/− mice to generate the double knockout mice, and the lesion size of aortic sinus was evaluated histologically between ESAM+/+ apoE−/− and ESAM−/− apoE+/+ mice. Plasma lipid profile was not affected by ESAM deficiency. However, the lesion size was markedly attenuated in ESAM−/− apoE−/− mice compared to ESAM+/+apoE−/− mice. The percentage of MOMA-2-stained area in the aortic sinus lesions was significantly smaller in ESAM-/-apoE−/− mice than in ESAM+/+apoE−/− mice, suggesting that ESAM deficiency reduced the macrophage infiltration in the atheroma. To clarify the mechanism for the reduced macrophage content in the plaque, in vitro adhesion- and transendothelial migration assays were performed between cultured endothelial monolayers and monocyte/macrophage cell line THP-1 cells utilizing siRNA-mediated knockdown of ESAM. These assays revealed that ESAM deficiency in endothelial cells resulted in decreases in monocyte adhesion to the endothelial cells as well as transendothelialmigration. THP-1 cells did not express ESAM, but directly bound to the recommbinant ESAM protein-coated culture plates.

Conclusion
: ESAM modulates macrophage infiltration into the atheroma through interaction with unidentified ligand(s) on monocytes. ESAM inactivation can reduce susceptibility to atherosclerosis.
Abstract 3946: Molecular Identification and Characterization of a Novel Gene that Regulates Endothelial Cell Apoptosis and Angiogenesis
Authors: Ikeda Koji , Koide Masahiro , Uraoka Maki , Nakagawa Yusuke , Nakano Ritsuko , Matsubara Hiroaki
Published: July 2021

Endothelial apoptosis is a pivotal process for angiogenesis during embryogenesis as well as postnatal life. Here, we identified and characterized a novel gene (ARIA) that regulates endothelial apoptosis and angiogenesis. ARIA was expressed in a variety of human cultured endothelial cells as well as in blood vessels of e9.5d mouse embryos, suggesting its role in angiogenesis. Amino acid sequence analysis revealed that ARIA is a novel membrane protein with no homology to conserved domain structures reported before. Knockdown of ARIA in HUVEC using siRNA resulted in significant reduction of endothelial apoptosis. In contrast, ARIA-knockdown did not affect either endothelial cell migration or proliferation. Among factors associated with apoptosis, inhibitor of apoptosis (cIAP)-1 and cIAP-2 protein expression was significantly increased by ARIA-knockdown whereas their mRNA level was not changed. cIAP-1 and cIAP-2 play as ‘gardians’ of the cell death machinery by directly bind to and neutralize caspases as well as by modulate death signaling of TNF receptors. Simultaneous knockdown of cIAP-1 and cIAP-2 abolished the anti-apoptotic effect of ARIA-knockdown. On the other hand, yeast two-hybrid screening using the putative intracellular domain of ARIA as the bait identified the 20S proteasome α subunit-7 as a binding partner of ARIA. Proteasomal inhibition significantly increased cIAP-1 and cIAP-2 expression in HUVEC, indicating their proteasomal degradation in endothelial cells. ARIA-knockdown cancelled this effect of proteasomal inhibition. ARIA-knockdown also mimicked proteasomal inhibition with respect to the expression and subcellular localization of cIAP-1 and cIAP-2 in HUVEC. These results strongly suggest that ARIA plays crucial roles in the proteasomal degradation of cIAP-1 and cIAP-2 in endothelial cells via its interaction with the 20S proteasome α subunit-7. Furthermore, in vivo angiogenesis assessed by Matrigel-plug assay was significantly enhanced by ARIA-knockdown. Taken together, ARIA is a novel factor regulating endothelial apoptosis and angiogenesis through modulating proteasomal degradation of cIAP-1 and cIAP-2. Inhibition of ARIA could be a novel target for developing the better therapeutic angiogenesis.
Abstract 5248: Direct Cardiac Effect and Cellular Mechanism of Angiotensin-(1–7) in Heart Failure
Authors: Cheng Che-Ping , Zhou Peng , Cheng Heng-Jie , Cross Michael , Little William C
Published: July 2021

Angiotensin-(1–7) {A-(1–7)} plays an important role in counteracting various actions of angiotensin II (AII) in the heart. However, its effect on cardiac contractility remains controversial. We previously demonstrated that in heart failure (HF), AII decreases left ventricle (LV) contractility. Whether A-(1–7) may antagonize AII-induced cardiac depression, thereby contributing to the beneficial actions of angiotensin-converting enzyme inhibitor (ACEI) therapy in HF, remains undefined. We assessed the hypothesis that A-(1–7) may produce positive modulation on Ca
2+
current (I
Ca,L
) and increase LV and myocyte contractility via A-(1–7) receptors, acting through nitric oxide (NO)/bradykinin (BK)-mediated mechanism. We measured LV contractility changes after A-(1–7) (650 ng/kg, iv), which produced more than a 20-fold increase in plasma A-(1–7) levels, mimicking the elevations caused by ACEI in HF patients, and compared myocyte contractile and I
Ca,L
responses to A-(1–7) (10
−5
M) in 12 rats with isoproterenol (ISO)-induced HF (3 months after 170 mg/kg sq for 2 days). LV contractility was measured by pressure-volume analysis. I
Ca,L
was measured using whole-cell voltage clamp technique. In additional contractile studies, myocytes were pretreated to inhibit either NO synthase (L-NAME, 10
−5
M), BK (HOE 140, 10
−6
M) or A-1–7 receptor (D-Ala
7
]-A-(1–7), 10
−5
M) followed with A-(1–7) exposure. Compared with baseline
,
after A-(1–7), E
ES
(44%, 1.1 vs 0.74 mmHg/μl) and M
sw
(37%, 91.2 vs 66.5 mmHg) were increased, indicating enhanced LV contractility. In HF myocytes, A-(1–7) increased myocyte percent shortening (28%, 7.3% vs 5.7%), the velocity of cell contraction (31%, 110.4 vs 84.2 μm/sec) and relengthening (41%, 71.8 vs 51.1 μm/sec) accompanied by significantly-increased peak I
Ca,L
(21%, 6.3±0.2 vs 5.2±0.2 pA/pF). L-NAME increased, HOE 140 decreased, and A-(1–7) receptor blockade prevented myocyte contractile responses to A-(1–7). In HF, clinically-relevant concentrations of A-(1–7) counteracted AII-induced cardiac depression, increased I
Ca,L
, and produced positive inotropic effects in both LV and myocytes. These effects are coupled with A-(1–7) receptors and involve activation of NO/BK pathways.
Abstract 5290: Cypher Long but not Short Isoform Specific Knockout Mice Result in Cardiac Signaling Defects and Dilated Cardiomyopathy
Authors: Cheng Hongqiang , Zheng Ming , Sheikh Farah , Ouyang Kunfu , Cui Li , Gu Yusu , Dalton Nancy , Chen Ju
Published: July 2021

Our previous studies have demonstrated that Cypher, a PDZ-LIM protein localized at the Z line, plays a pivotal role in heart function. We recently identified long and short splice isoforms of Cypher, which are characterized by the presence and absence of LIM domains, respectively. The LIM domain of Cypher is thought to be involved in signaling, based on its ability to directly interact with signaling proteins. In human patients with dilated cardiomyopathy (DCM) we discovered Cypher gene mutations, which affect either long or short isoform or both isoforms. However, the precise molecular mechanisms underlying the role of Cypher isoforms in DCM remain unclear. To determine the role of Cypher isoforms in cardiac signaling and disease
in vivo
, we generated two Cypher isoform specific knockout mice. Selective ablation of Cypher long isoforms in mice resulted in partial neonatal lethality. However, hearts from viable Cypher long isoform deficient mice displayed Z line abnormalities and decreased cardiomyocyte widths, which resulted in a progressive form of DCM, characterized by fibrosis, calcification and lethality. The effects on cardiac function and disease observed in long-isoform specific Cypher knockout mice were preceded by significant decreases in cardiac protein kinase C and extracellular signal-regulated kinase signaling. These results are in contrast to Cypher short isoform deficient mice, which were viable with no overt cardiac morphology and signaling abnormalities. These results reveal distinct functional roles for Cypher isoforms in the heart as well as shed light into the molecular mechanisms underlying dilated cardiomyopathy.
Abstract 5314: Domain
2114–2149
Within RyR2 Plays a Key Role for Correcting Channel Disorder in Catecholaminergic Polymorphic Ventricular Tachycardia (CPVT)
Authors: Yamamoto Takeshi , Yano Masafumi , Suetomi Takeshi , Ono Makoto , Xu Xiaojuan , Uchinoumi Hitoshi , Tateishi Hiroki , Okuda Shinichi , Kobayashi Shigeki , Matsuzaki Masunori
Published: July 2021

We recently identified the K201(JTV519)-binding site as domain2114–2149 of the ryanodine receptor (RyR2), and the binding of K201 to this domain corrects the defective inter-domain interaction between N-terminal (0–600) and central regions (2000–2500) of RyR2 in pacing-induced failing hearts. Here, we further investigated the role of the domain peptide2114–2149 (DP
2114–2149
) on the Ca
2+
release in human CPVT-associated RyR2
R2474S/+
knock-in (KI) mice model. The KI mice revealed no structural or histological abnormality in hearts, and also had no contractile or relaxation dysfunction at rest. In all KI mice (n= 6), however, bi-directional ventricular tachycardia (VT) was observed after exercise with treadmill (Ex:6/6), but not observed in wild-type (WT) mice (Ex:0/7 ). In saponin-permeabilized cardiomyocytes from WT and KI mice, line scan images in cardiomyocytes were obtained to measure local Ca
2+
release events using a confocal microscopy with Rhod-2; [Ca
2+
] was buffered at 30 nM by 0.5 mM EGTA. In KI, the frequency of Ca
2+
sparks (SpF: s
−1
·100μm
−1
) was much more increased in response to cAMP (0.1–1 μM) than in WT (KI:19.2±0.4 vs WT:14.9±0.4; at [cAMP]= 0.3 μM , p<0.01). DP
2114–2149
abolished the cAMP-induced increase in SpF in KI. Sarcoplasmic reticulum (SR) vesicles were isolated from WT and KI hearts (n=4), then RyR2 was fluorescently labeled with methylcoumarin acetamido (MCA) using DP
2460–2495
(DPc10), which harbors the same mutation site in CPVT as KI mice;R2474S, as a site-directing carrier. Addition of cAMP (1 μM) to the SR from KI (but not WT) mice reduced stabilizing interactions between the N-terminal
1–600
and central domains
2000–2500
(i.e. domain unzipping), as assessed by the quenching of the MCA fluorescence by a large-size fluorescence quencher (Kq: 1.16 in KI: 1.10 in WT). Interestingly, DP
2114–2149
corrected this domain unzipping in KI (Kq:1.13), although DP
2114–2149
had no effect on the cAMP-induced increase in Ser2808 phosphorylation. In conclusion, RyR2 mutation seen in human CPVT may causatively induce hyper-activated channel gating in response to PKA phosphorylation, via defective inter-domain interaction. Correction of the defective inter-domain interaction may be a new therapeutic strategy against CPVT.
Abstract 5340: Cardiac Resynchronization Therapy Corrects Dyssynchrony-induced Regional Gene Expression Changes on a Genomic Level
Authors: Barth Andreas S , Aiba Takeshi , Halperin Victoria , DiSilvestre Deborah , Khalid Chakir , Dimaano Veronica L , Yu Wayne , Abraham Theodore , Kass David , Tomaselli Gordon F
Published: July 2021

Purpose:
Cardiac Resynchronization Therapy (CRT) improves symptoms and reduces mortality in patients with heart failure (HF). To characterize the molecular processes associated with functional improvement in CRT, we used a genomic approach in a large animal HF model.

Methods:
After creation of a left bundle branch block (LBBB), dogs in the HF group were subjected to either rapid atrial pacing with 200 bpm for 6 weeks (dyssynchronous HF, DHF, n=10), or 3 weeks of atrial pacing followed by 3 weeks of biventricular stimulation at 200bpm (CRT, n=9). Control animals without LBBB were not paced (NF, n=11). After 6 weeks, RNA from anterior and lateral regions of the LV was hybridized onto canine 44K arrays. Statistical Analysis of Microarrays (SAM) was used for data analysis.

Results:
Echocardiographically, CRT led to a significant increase in stroke volume (+27%, p=0.03) which translated into a non-significant increase in EF (DHF 25±4%; CRT 31±3% (p=0.15); NF 67±3%). A multiclass analysis of NF, DHF and CRT animals identified 1050 differentially expressed transcripts between anterior and lateral walls with a false discovery rate of 5%. For all these transcripts, dyssynchrony-induced expression changes were reversed by CRT to levels of NF hearts. As a result, CRT samples clustered with NF rather than DHF samples. Of particular interest were genes encoding for signal transduction pathways and contractile processes.

Conclusions:
By using a whole genome approach, we demonstrate a profound effect of electrical activation on the regional cardiac transcriptome. This is the first study showing that dyssynchrony-induced gene expression changes can be corrected by CRT on a genome-wide level.