bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1712: The P2Y
12
Antagonist Cangrelor and the GP IIb/IIIa Blocker Abciximab Reduce Platelet-Mediated Myocardial Injury After Ischemia and Reperfusion
Authors: Barrabes Jose A , Inserte Javier , Mirabet Maribel , Quiroga Adoracion , Hernando Victor , Figueras Jaume , Garcia-Dorado David
Published: July 2021

Objective:
Platelets activated during experimental acute myocardial infarction (AMI) contribute to myocardial injury. We aimed to investigate whether platelets from patients with AMI increase myocardial damage after transient ischemia in isolated rat hearts and the modification of this effect by the P2Y
12
receptor antagonist cangrelor and the GPIIb/IIIa receptor blocker abciximab.

Methods:
Platelets were obtained from 9 AMI patients (7 thrombolyzed, all on aspirin) within 24 h after symptom onset. Incubation with 100 μM cangrelor or 50 μg/ml abciximab resulted, respectively, in 78 ± 4 and 90 ± 2% inhibition of aggregation (optical aggregometry). Isolated rat hearts (four simultaneous experiments per patient) were subjected to 40 min of global ischemia and 60 min of reperfusion. Hearts received no additional intervention (Control) or were infused during the 5 min prior to ischemia with platelets (22.5x10
6
/min), either untreated or treated with cangrelor or abciximab.

Results:
P-selectin expression (flow cytometry) in isolated platelets before infusion was 31 ± 3% (P = NS between groups). Platelets augmented myocardial injury, as demonstrated by worse left ventricular developed pressure (LVDevP), higher left ventricular enddiastolic pressure (LVEDP) and coronary resistance, and greater LDH release and infarct size (TTC staining), and both cangrelor and abciximab greatly attenuated these effects (Table

).

Conclusions:
Activated platelets from patients with AMI increase myocardial injury after ischemia and reperfusion, and cangrelor and abciximab attenuate this effect. The results support the notion that very early antiplatelet treatment may increase myocardial salvage by direct effects on the microcirculation in these patients.
Abstract 1749: Endothelial Progenitor Cells and Vascular Endothelial Function Follow a Parallel Circadian Pattern
Authors: Mheid Ibhar Al , Aznaouridis Konstantinos , Patel Riyaz , Shirazi Farheen , Sutcliffe Diane J , Taylor W. R , Alexander R. W , Quyyumi Arshed
Published: July 2021

Background: Endothelial Progenitor Cells (EPCs) are mobilized in response to endothelial injury and their counts correlate with endothelial function and cardiovascular risk burden. This rare cell population can be characterized by either flow cytometric analysis or mononuclear cell culture and colony counting, however, the 24-hour circadian variation and inter-relation of EPC measures with endothelial function remain unknown. We hypothesized that EPC levels and endothelial function will have a similar circadian variation.

Methods: In 12 healthy subjects (8 men, 42 ± 18 yrs), we investigated the circadian variation of: 1-circulating cells positive for:

CD34,

CD133 andCD34,

the number of colonies formed by culturing mononuclear cells utilizing a 7-day assay and

flow-mediated vasodilation utilizing brachial artery reactivity at 8am, noon, 4pm, 8pm, midnight, 4am and 8am.

Results: ANOVA for repeated measures over 24 hours indicated significant changes in the number of colony counts (p < .001, highest at midnight). Similarly, flow-mediated vasodilation was highest at midnight (p < .001). In contrast, CD34+ and dual positive CD34+ /CD133+ cell counts peaked a little earlier at 8 pm (8 pm vs.8 am p = .07 and .01 for paired t-tests, respectively). (Figure

)

Conclusion: Endothelial function and EPCs estimated as colony forming units peak at midnight and are lower at other times of the day. The highest counts of circulating progenitors are seen at 8pm. There is a parallel change in circulating EPCs and vascular endothelial function during a 24-hour period, with lower levels in the morning hours.
Abstract 1771: Serum And Glucocorticoid-induced Kinase-1 (SGK-1) Dynamically Regulates Cardiac Fibrosis In A CTGF- Independent Manner
Authors: Rosenberg Michael A , Das Saumya , Pinzon Pablo Quintero , Xiao Chunyang , Banerjee Sucharita , Rosenzweig Anthony
Published: July 2021

Intro: We previously reported that the mineralcorticoid-responsive kinase, SGK-1, is activated in cardiac hypertrophy and regulates cardiomyocyte (CM) growth and survival. SGK-1 has also been implicated in renal fibrosis through transcriptional induction of connective tissue growth factor (CTGF). In this study, we examined the role of CM SGK-1 activation in the development of cardiac fibrosis.

Methods: We studied histology and electrophysiology in transgenic mice with cardiac-specific overexpression of constitutively-activated (SGK-CA) and kinase-dead (SGK-KD) SGK-1 protein. Protein and RNA expression were measured by Western blot and QRT-PCR, respectively, and SGK-KD mice were studied following transverse aortic constriction (TAC).

Results: EKG analysis revealed QRS prolongation in SGK-CA mice compared with wild type (WT) littermates (22.3±6.6 ms vs. 15.7±4.3 ms, p<0.05), which correlated with increasing age (R
2
0.44, p<0.05). Histologically, SGK-CA mice displayed increased fibrosis on Mason’s trichrome stain compared with WT mice. Protein expression of connexin43 (Cx43) was decreased 33% in SGK-CA ventricles (p<0.05), without a change in Cx43 RNA, consistent with post-transcriptional downregulation secondary to fibrosis. TGF-β RNA expression was increased in SGK-CA mice 1.44 fold (p<0.05), as was the fibrosis marker fibronectin (1.22 fold, p<0.05), while CTGF RNA expression was not significantly different between SGK-CA mice and wild type littermates. Following TAC, the SGK-KD mice displayed decreased cardiac fibrosis compared with TAC WT mice. Paradoxically, SGK-KD had increased expression of CTGF RNA vs. WT both at baseline (1.26 fold, p<0.05), as well as after TAC (1.52 fold, p<0.05), with both groups having > 3 fold increase in CTGF RNA after TAC. TGF- β RNA expression also increased about 1.5 fold in each group with TAC, although there was no significant difference between SGK-KD and WT mice either at baseline or after TAC.

Conclusion: Cardiomyocyte SGK-1 activation appears to play a dynamic role in the development of cardiac fibrosis as well as subsequent conduction disease. Our finding of a disconnect between fibrosis and induction of CTGF or TGF- β in SGK mice suggests that it works at least in part through independent pathways.
Abstract 1772: Cardiomyocyte Damage-Released S100A1 Protein Evokes A Proinflammatory Phenotype In Cardiac Fibroblasts
Authors: Rohde David , Boerries Melanie , Nicole Herzog , Qiu Gang , Ehlermann Philipp , Nawroth Peter P , Katus Hugo A , Bierhaus Angelika , Peppel Karsten , Most Patrick
Published: July 2021

Background:
S100A1, a cardiomyocyte specific inotropic calcium sensor protein, is released from infarcted human myocardium in the extracellular environment and circulation, reaching peak serum levels (1–2 μM) 8–9 hours after clinical onset. As growing evidence indicates that S100 proteins can act as pre-existing danger signals triggering the innate immune system into action upon release from injured host cells, we hypothesized that damage-released S100A1 can act as a cardiac danger signal alerting innate immune cells.

Methods and Results:
Here we report for the first time that necrotic cardiomyocytes release S100A1 protein in vitro, which is exclusively internalized by cardiac fibroblasts (CFs) in a clathrin- and caveolin-independent manner as shown by IF. Internalized S100A1 specifically activated MAPKs/SAPKs (p38, ERK1/2 and JNK) resulting in nuclear translocation of p65 (NF-kB) as assessed by Western blotting, EMSA and IF. In turn, S100A1 triggered an inflammatory gene program in CFs including enhanced expression of adhesion molecules, integrins, chemokines and cytokines including I-CAM, V-CAM, CD11b/18, IL1-alpha, MCP-1, TNF-alpha, SDF-1 among others as obtained by RT-PCR, Western blotting and ELISA. This resulted in enhanced chemoattraction and adhesion of monocytotic and stem cells to S100A1-activated CF as shown by Boyden-chamber and adhesion assays. In line with their proinflammatory transition, S100A1-activated CFs exhibited decreased collagen-1/-3 expression and de-novo collagen production, enhanced collagenolytic MMP-9 abundance and activity and increased levels of the antiangiogenic matricellular factor thrombospondin-2 reflecting extracellular matrix net degradation. Importantly, the immun-modulatory and antifibrotic actions of S100A1 protein in vitro were restricted to CFs, RAGE independent and occurred at concentrations (0.1–1 μM) that were found in patients after AMI.

Conclusion:
Our in vitro results indicate that S100A1 has the properties of a pre-exisiting endogenous cardiomyocyte danger signal transforming cardiac fibroblasts into immunmodulatory cells that might recruit innate immune cells to the site of cardiac injury and link cardiomyocyte damage to post-MI inflammation.
Abstract 1781: Activation of a Protein Deacetylase, SIRT1, by Resveratrol Suppresses Progression of Heart Failure and Improves Survival by Up-Regulation of Mn-SOD
Authors: Tanno Masaya , Kuno Atsushi , Miki Takayuki , Miura Tetsuji , Horio Yoshiyuki , Shimamoto Kazuaki
Published: July 2021

[Purpose] SIRT1, a protein deacetylase, induces cellular resistance against oxidatnt stress by deacetylating forkhead transcription factors and p53. However, the physiological significance of the protection and the role of endogenous free radical scavengers have not been clarified. Here, we examined involvement of Mn-SOD in the SIRT1-mediated protection and possible benefit of a SIRT1 activator, resveratrol (RSV), for suppressing progression of heart failure. [Methods and Results] First, capacity of SIRT1 to prevent oxidant-induced apoptosis and its mechanism were examined in cardiomyocytes. Apoptosis induced by antimycin A (AA, 30 μM), an oxidant stressor, was suppressed by transfection with wild-type SIRT1 (WT) (4.4±0.7%) compared with the control (40.4±5.5%), while deacetylase-inactive SIRT1 (H355Y) was not protective (38.0±8.1%). WT, but not H355Y, increased Mn-SOD by 3.7 fold, and knockdown of Mn-SOD by siRNA abolished both up-regulation of Mn-SOD and protection against AA. RSV (100 μM) significantly reduced acetyl-histone H3 level and mimicked the effect of WT on AA-induced apoptosis (26.5±4.8% vs. 11.5±1.1%). These effects were attenuated by siRNA-mediated knockdown of SIRT1, indicating that SIRT1 is required for the protection. Second, the effect of SIRT1 activation by RSV on heart failure was examined in TO-2 cardiomyopathic hamsters. Treatment with RSV (4 g/kg in chow) was commenced at the age of 6 weeks. Echocardiography was performed at 25 weeks, and determination of mRNA levels and immunohistochemistry were performed at 35 weeks. Compared with those in controls, LVEF was significantly better preserved (34.3±2.2% vs. 27.9±1.6%, p=0.02), Mn-SOD protein was increased by 32% (p=0.04), BNP mRNA level was reduced by 31% (p<0.01) and interstitial fibrosis detected by fibronectin staining was decreased by 53% (p=0.02). Furthermore, Kaplan-Meier analysis revealed that RSV significantly improved the survival of TO-2 hamsters from 7% to 27% during a 51-week follow-up period (n=29, Hazard ratio = 0.417, χ2 = 4.83, p=0.028 vs. control). [Conclusion] Progression of heart failure and death can be suppressed by activation of SIRT1 by the use of RSV, this being attributable to suppression of cardiomyocyte apoptosis by up-regulation of Mn-SOD.
Abstract 1783: Pro-Apoptotic Bnip3 Mediates Permeabilization of Mitochondria and Release of Cytocrome c via a Novel Mechanism
Authors: Quinsay Melissa N , Rikka Shivaji , Sayen M Richard , Molkentin Jeffery D , Gottlieb Roberta A , Gustafsson Asa B
Published: July 2021

Bnip3 is a member of the BH3-only subfamily of pro-apoptotic Bcl-2 proteins and is associated with mitochondrial dysfunction and cell death in the myocardium. The pro-apoptotic Bcl-2 proteins mediate mitochondrial dysfunction independent of the mitochondrial permeability transition pore (mPTP). However, Bnip3 has been reported to mediate cell death via the mPTP. In this study, we investigated the mechanism(s) by which Bnip3 causes mitochondrial dysfunction. Using a mitochondrial swelling assay to assess pore opening, we found that addition of 200 microM Ca2+ to mitochondria isolated from rat hearts induced rapid swelling of mitochondria and release of cytochrome c (cyto c). Bnip3 also induced mitochondrial swelling and cyto c release, but always at a slower rate and to a greater degree, suggesting that Bnip3 mediates swelling via a different mechanism. Cyclosporin A (CsA), an inhibitor of mPTP opening, prevented Ca2+-induced swelling and cyto c release, but had no effect on Bnip3. Another BH3-only protein, tBid, caused release of cyto c but failed to induce swelling of mitochondria. Interestingly, Bnip3, but not Ca2+ and tBid, induced release of the matrix protein MnSOD. Cyclophilin D (cycD) is an essential component of the mPTP and heart mitochondria isolated from cycD−/− mice were resistant to Ca2+-, but not to Bnip3-induced swelling and cyto c release. Also, tBid caused cyto c release without mitochondrial swelling in the absence of cycD. To further explore the mPTP as a downstream effector of Bnip3-mediated cell death, we assessed cell death in mouse embryonic fibroblasts (MEFs) isolated from wild type (wt) and cycD−/− mice. Infection with an adenovirus expressing Bnip3 caused significant cell death in wt (52.8±1.8%) and cycD−/− (61.8±6.1%) MEFs as measured by LDH release. In addition, both Bnip3 and opening of the mPTP have been reported to initiate upregulation of autophagy. Monitoring of GFP-LC3 incorporation into autophagosomes by fluorescence microscopy revealed that Bnip3 infection induced autophagy in wt (86.5±6.6%) and cycD−/− (96.4±1.4%) MEFs (n=3, p<0.05). Thus, these studies suggest that Bnip3 mediates permeabilization of the inner and outer mitochondrial membranes via a novel mechanism that is different from other BH3-only proteins.

This research has received full or partial funding support from the American Heart Association, AHA National Center.
Abstract 1784: Mir-199a Is A Key Regulator Of Hif-1alpha And An Inhibitor Of Hypoxia-induced Apoptosis
Authors: Rane Shweta , He Minzhen , Sayed Danish , Hong Chull , Abdellatif Maha
Published: July 2021

MicroRNAs (miRNA) are touted to be fundamental posttranscriptional regulators of gene expression. They are differentially regulated during health and disease states with a major impact on cellular functions. We found that miR-199a is upregulated (4.8 ± 1 fold) during compensatory cardiac hypertrophy, returning to basal levels in the failing heart, and is virtually undetectable during cardiac ischemia. The response of miR-199a to ischemia is rapid through posttranscriptional inhibition of its processing. To determine its targets we analyzed myocytes overexpressing miR-199a by using an antibody array analysis, the outcome of which unequivocally indicated an antiapoptotic function of this miRNA. Target prediction software revealed hypoxia-inducible factor 1 alpha (Hif-1α) as one of its potential targets. We confirmed this by protein expression, luciferase-miR-199a target reporter, and recombinant Hif-1α gene expression under hypoxic conditions in cardiac myocytes. The results show that miR-199a inhibited hypoxia-induced Hif-1α > 70%, and vice versa, knockdown of miR-199a by a miR-199a ‘eraser’ resulted in its upregulation (1.8 fold). Hif-1α, which is undetectable during normoxia and induced by hypoxia, mediates hypoxia-induced apoptosis through a p53-dependent pathway. MiR-199a overexpression in myocytes exposed to anoxia or ischemia resulted in inhibition of Hif-1α expression, as well as, >80% inhibition of the increase in p53. Moreover, miR-199a inhibited hypoxia-induced expression of caspase-6, caspase-9, caspase-12, and caspase-3 activity (~60%). To determine if the protective effect of miR-199a was indeed mediated through inhibition of Hif-1α, we co-delivered a miR-199a-indepndent Hif-1α construct to the myocytes. This resulted in complete reversal of the miR-199a effect on anoxia-induced p53 expression and its downstream caspase effectors. Thus, our results indicate that downregulation of miR-199a by hypoxia is necessary for enhancing expression of Hif-1α and its downstream apoptosis effector p53. Moreover, we show that replenishing miR-199a protects the myocytes against ischemia- or hypoxia-induced apoptosis.
Abstract 1823: Inhibition of Histone Deacetylases Accelerates Unloading-Induced Cardiac Atrophy
Authors: Blagg Andrew G , Rothermel Beverly A , Hill Joseph A
Published: July 2021

Background:
Mechanical unloading of the diseased ventricle has emerged as an important treatment strategy in advanced heart failure. In rare instances, dramatic improvements in ventricular performance are seen, yet little is known regarding underlying mechanisms. Histone deacetylases (HDACs) are implicated in the control of cell growth and remodeling. Recent studies have demonstrated that HDAC inhibition (HDACi) suppresses pathological cardiac hypertrophy and attenuates re-induction of the fetal gene program. We hypothesized that HDACi would accelerate unloading-induced cardiac atrophy.

Methods:
Mechanical unloading of murine hearts was accomplished by heterotopic transplantation into the abdomen of same-strain recipients (7 days). Two days prior to surgery, mice were randomized to daily sq injections of either trichostatin A (TSA, 1 mg/kg) or vehicle. Left ventricular (LV) cardiomyocyte area was measured from H&E sections (100 myocytes/heart), and LV transcript abundance was measured by quantitative real-time RT-PCR. Native hearts from recipient mice served as controls.

Results:
Consistent with a role for HDACs in cardiac atrophy, the protein abundance of HDAC-1 (54%; p<0.05), -2 (52%; p<0.01) and -3 (40%; p<0.05) were significantly increased in untreated donor hearts. Unloading-induced cardiomyocyte atrophy was accelerated in hearts transplanted into TSA-treated mice (25±4% atrophy, TSA vs 13±1% atrophy, vehicle; p<0.05). Remarkably, TSA treatment did not blunt (p=NS) the dramatic (72-fold, p<0.05) up-regulation of fetal
β
MHC seen in vehicle-treated hearts, despite significant increases in the rate of atrophy development. The cyclin dependent kinase inhibitors (CDKIs) p21(Cip1/Waf1) and p27(Kip1), which are known to negatively regulate cardiac hypertrophy, were each increased (p21, 45%, p<0.1; p27, 31%, p<0.1) in TSA-treated hearts relative to control.

Conclusions:
HDAC inhibition elicits a bona fide cardiomyocyte atrophy response during ventricular unloading in association with up-regulation of CDKI transcript expression. HDAC inhibition, however, does not blunt re-activation of the fetal gene program. These findings may have important implications for heart failure patients undergoing LV assist device support.

This research has received full or partial funding support from the American Heart Association, AHA South Central Affiliate (Arkansas, New Mexico, Oklahoma & Texas).
Abstract 1839: MicroRNA-499 Promotes the Differentiation of Cardiac Progenitor Cells into Myocytes
Authors: Hosoda Toru , Urbanek Konrad , Carvalho Adriana Bastos , Bearzi Claudia , Bardelli Silvana , Rizzi Roberto , D’Amario Domenico , Carrillo-Infante Cynthia , Zheng Hanqiao , Quaini Federico , Rota Marcello , Anversa Piero , Leri Annarosa
Published: July 2021

Myocardial regeneration mediated by cardiac progenitor cells (CPCs) results in the partial recovery of the infarcted heart but the newly formed myocytes within the necrotic tissue have fetal-neonatal characteristics. In contrast, CPC activation in the remote viable myocardium results in the formation of mature myocytes, suggesting that CPC differentiation is conditioned by the surrounding cells. Thus, the hypothesis is raised that microRNAs (miRs) that are highly expressed in myocytes and are absent in CPCs, may translocate through gap junctions to adjacent CPCs promoting their differentiation. By employing miR array and Q-RT-PCR, miR-499 was found to be ~500-fold more expressed in myocytes than CPCs. Additionally, we demonstrated that miR-499 translocates from neighboring cells to CPCs through the formation of gap junctions. The translocated miR-499 was functional and repressed the expression of target genes. Among 200 putative targets of miR-499, we have elected to study Sox6 and Rod1. The validation of these putative miR-499-targets was obtained by reporter assays; cells transfected with miR-499 together with plasmids carrying luciferase and the 3′-UTR region of Sox6 or Rod1 show the expected decrease in luciferase activity. Transcripts of Sox6 and Rod1 were measured by Q-RT-PCR in myocytes and CPCs; Sox6 mRNA was 2-fold higher and Rod1 mRNA was 98% lower in myocytes than CPCs. However, the protein levels of Sox6 and Rod1 were significantly lower in myocytes than CPCs suggesting that miR-499 promotes degradation and/or inhibition of translation of these target genes. To document miR-499 function, CPCs were transfected with a miR-499-expression vector and cell proliferation and differentiation were evaluated 3 days later. BrdU incorporation decreased 60% and the cells displayed a marked upregulation of the myocyte-specific transcription factors Nkx2.5 and MEF2C. Similar results were obtained when Sox6 and Rod1 were selectively blocked with siRNA. In both cases, the number of Nkx2.5- and MEF2C-positive cells increased 2–3-fold. Thus, our data indicate that miR-499 translocates via gap junction from myocytes to CPCs where miR-499 is a crucial modulator of the differentiation of CPCs into cardiomyocytes through the repression of Sox6 and Rod1.
Abstract 1890: Depletion of Transplanted Pro-Angiogenic Cells by a Suicide Approach Discloses a Crucial Role of Sustained Incorporation for Cardiac Repair after Myocardial Infarction
Authors: Yoon Chang-hwan , Ziebart Thomas , Trepels Thomas , Wietelsbacher Astrid , Braun Thomas , Stein Stefan , Grez Manuel , Ihling Christian , Urbich Carmen , Zeiher Andreas , Dimmeler Stefaine
Published: July 2021

Endothelial progenitor cells improve neovascularisation of ischemic tissue by a broad repertoire of potential mechanisms. While initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. The question remains to what extent a physical incorporation is contributing to the beneficial effects of cell therapy. Pro-angiogenic cells were cultivated from peripheral blood mononuclear cells under conditions favouring endothelial commitment (“early EPC”) and were transduced with a lentiviral vector encoding thymidine kinase (TK) under the control of constitutively active promoter. TK activates the pro-drug gancyclovir (GV) to induce toxicity in the target cell. Injection of cells in mice after acute myocardial infarction (MI) significantly improved left ventricular ejection fraction (EF) as assessed by MRI and echocardiography up to 4 weeks compared to control mice. However, depletion of the TK-expressing cells by GV treatment at 2 weeks after cell infusion was associated with deterioration of EF measured at 4 weeks (no cells:37 ± 5%;TK-cells:61 ± 5.6%,TK-cells + GV:43 ± 4.6%). Likewise, endsystolic volume was significantly reduced in mice injected with TK-cells but was increased by cell depletion with GV. Consistently, the increased capillary density in TK-cell-treated mice was significantly reduced by GV treatment (lectin
+
area: TK-cells: 11 ± 2.0, TK-cells+GV: 6.8 ± 2.5, p < 0.05). In order to examine the contribution of lineage-committed cells after MI, we selectively depleted KDR-positive cells by expressing TK under the control of the KDR-promoter. Injection of gancyclovir after 2 weeks in KDR-TK-cell treated mice also resulted in a deterioration of the EF measured at 4 weeks, however, the effect was less pronounced compared to the depletion of all cells by using the constitutively active promoter.

Conclusion: Depletion of infused cells 2 weeks after MI induced a decline of EF and capillary density. These data document that infused cells are physically incorporated weeks after transplantation. The contribution of specific cell lineages to the maintenance of functional improvement warrants further investigations.
Abstract 1964: Bone Morphogenetic Protein Antagonist “Protein Related to Dan and Cerberus” Promotes Differentiation of Embryonic Stem Cells to Atrial Cardiomyocytes
Authors: Yan Jingbo , Hu Jianyong , Mueller Iris I , Heaton William H , Wang Wan-Der , Dzhura Igor , Baudenbacher Franz , Knapik Ela W , Hatzopoulos Antonis K
Published: July 2021

The molecular factors that regulate cardiac differentiation have been extensively studied, yet, relatively little is known about how cardiomyocytes acquire atrial versus ventricular characteristics. Embryonic stem (ES) cells, which have the potential to differentiate to a wide array of distinct cell types, including most types of cardiovascular cells, offer a pertinent in vitro model to work out the molecular mechanisms of atrial specification and differentiation. We discovered that the secreted antagonist of BMP signaling, Protein Related to Dan and Cerberus (PRDC, also called Gremlin2) leads to a surge in cardiomyocytic differentiation when applied to mouse ES-derived cardiac progenitor cells. This property is unique to PRDC among tested BMP antagonists. Lineage expansion is restricted to cardiomyocytes, with the differentiation of endodermal, blood, endothelial and neuronal cells being unaffected. Using molecular and electrophysiological analyses, we show that PRDC-induced cardiomyocytes acquire atrial characteristics. Consistent with the in vitro results, we found that injection of PRDC mRNA into the developing zebrafish embryo leads to supernumerary contracting areas. The ectopic cardiomyocytes express atrial-, but not ventricular- specific cardiac genes. We determined that PRDC treatment induces the expression of COUP-TFII, a known transcriptional regulator of atrial differentiation, but suppresses Notch signaling. Inhibition of Notch is sufficient to induce atrial-specific genes; however, blocking Notch does not expand the cardiogenic fields. Taken together, our data suggest that antagonism of BMP and Notch signaling by PRDC is a critical early step in the specification, expansion and differentiation of atrial progenitor cells. This information might be relevant for treating atrial degeneration, as well as for understanding the etiology of atrial fibrillation.
Abstract 3408: Folate Suppresses Wnt/Beta-Catenin Inhibition of Cardiogenesis to Protect Against Induction of Congenital Heart Defects
Authors: Serrano Maria , Han Mingda , Huhta James C , Lastra-Vicente Rosana , Garita Barbara , Linask Kersti K
Published: July 2021

Lithium (Li), a mood stabilizing drug, and elevated homocysteine (HCy), a metabolite in the folic acid (FA) cycle, are linked to induction of human congenital heart defects. We determined noninvasively by echocardiography, that exposure of the mouse embryo by an i.p. injection to the dam of Li or HCy during
gastrulation
on embryonic day
E6.75
induces cardiac and valve defects. A
single dose
of 125 μl Li (25 mM) or HCy (75 μM) on E5.5 to 6.5 results in mouse embryonic lethality; on E6.75 to 7.0, both induce tricuspid and semilunar valve defects (Li: n=131 ; HCy: n=78). The tricuspid valve septal leaflet fails to delaminate, a characteristic of Ebstein’s Anomaly. Use of Li during human pregnancy has been associated with Ebstein’s Anomaly. Li mimics the Wnt/β-catenin (β-cat) signaling pathway by inactivating glycogen synthase kinase-3β. During chick cardiac specification, Li, Wnt 3A, or HCy exposure adversely affects chick cardiogenesis with severity of anomalies based on timing of early exposure. To initiate cardiogenesis, the secreted Wnt
antagonist Dickkopf-1
acts extracellularly on the endoderm to upregulate
Hex
, an inducer of cardiomyogenesis. Exposure of stages 3+/4− chick embryos to Li/Wnt3A/ HCys inhibits
Hex and Islet-1
gene expression in the cardiogenic crescent via an intracellular mechanism, thus augmenting inhibitory Wnt/β-cat signaling. FA deficiency leads to elevated HCys levels. We hypothesize that HCys/FA metabolism intersects with Wnt/β-cat signaling and that mechanistically FA supplementation acts by overriding Wnt/β-cat inhibition of gene expression in the embryonic heart fields that leads to cardiac defects. FA, known to protect against HCys-mediated neural tube defects, was tested for protective effects against Li/Wnt3A/HCy during cardiogenesis. With all three experimental exposures, FA addition results in reexpression of the cardiac inducers
Hex and Islet-1
at high levels in the chick heart fields. In the mouse, no valve defects were detected in HCys/FA exposed embryos (n=27). Regimen and concentration of FA supplementation necessary to fully rescue Li effects are being tested. In conclusion, folate supplementation potentiates the repression of Wnt/β-cat signaling and protects formation of heart and valve defects.

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 3420: Potential of Human Mesenchymal Stem Cells to Differentiate into Cardiomyocytes Depends on Donor Age and Donor Tissue
Authors: Van Tuyn John , Pijnappels Daniel A , Haeck Marlieke L , Knaän-Shanzer Shoshan , Scherjon Sicco , Goumans Marie-José , de Vries Antoine A , van der Laarse Arnoud , Schalij Martin J , Atsma Douwe E
Published: July 2021

Treatment of damaged myocardium using autologous adult stem cells is under intense investigation as this may regenerate lost myocardium, thereby improving cardiac function. Initial cardiac cell therapy trials using various adult stem cell types have shown beneficial effects. However in contrast to earlier animal studies no true, quantitative reconstitution of myocardium has been observed. We hypothesized that the cardiomyogenic potential of stem cells depends on donor age and cell source. We used an in vitro differentiation protocol that employs co-culture of enhanced green fluorescent protein-labeled human mesenchymal stem cells (hMSCs) with neonatal rat cardiomyocytes (nrCMCs) for 13 days, without additional chemical or mutagenic stimulation. hMSCs were either derived from adult patients, or from human fetal tissue (gestational age 18 –22 weeks) or post natally collected umbilical cord blood. Differentiation of hMSCs was assessed by the presence of a properly developed sarcomere using immunological labeling for cardiac troponin I, myosin heavy chain and actinin. Functionality of newly differentiated cardiomyocytes was assessed by the presence of a functional calcium transient in conjunction with myocyte contraction. Mesenchymal stem cells isolated from clinically relevant sources (bone marrow, fat, and synovium) of adult patients are unable to adopt a cardiomyocyte phenotype upon co-incubation with nrCMCs. In contrast, MSCs from fetal donor material all give rise to functional cardiomyocytes, although with varying efficiency (umbilical cord blood: 6.8%±3.0, bone marrow: 0.4%±1.4, liver: 1.8%±0.8, lung: 1.4%±1.3, amnion: 9.3%±5.0 [mean±SD, n=3]). Using short hairpin RNA-mediated knockdown of connexin 43 (Cx43) gene expression, we found that sarcomeric development (organization) depends on the presence of Cx43 in the differentiating stem cells. Both donor age and the tissue source of human MSCs critically influence their cardiomyogenic differentiation potential. These results suggest that these non-embryonic, fetal cells may potentially be clinically useful for cardiac cell therapy.
Abstract 3444: Arachidonic 12-lipoxygenase Promotes the Development of Cardiac Fibrosis and Heart Failure via Induction of Monocyte Chemoattractant Protein-1
Authors: Kayama Yosuke
Published: July 2021

To elucidate the molecular mechanisms of heart failure, we examined expression of 8800 genes in the heart of hypertensive heart failure model (Dahl salt-sensitive rats). DNA chip analysis revealed that 12-lipoxygenase (12-LOX) was markedly upregulated in the failing heart. 12-LOX is a key enzyme of the arachidonic cascade that metabolizes eicosanoid. Until recently, 12-LOX has been reported to play an important role in the development of atherogenesis, diabetes, and neurogenerative disease. However, the role of 12-LOX in heart failure has not been examined. To determine whether increased expression of 12-LOX causes heart failure, we established transgenic mice that overexpress 12-LOX only in cardiomyocytes. Echocardiogra-phy showed that 12-LOX transgenic mice developed systolic dysfunction from as early as 16 weeks old. Histological analysis revealed that cardiac fibrosis was increased in 12-LOX transgenic mice with advancing age, which was associated with infiltration of macrophages. Consistent with these observations, cardiac expression of monocyte chemoattractant protein-1 (MCP-1) was upregulated in 12-LOX transgenic mice compared to those of wild-type mice. In vitro experiments demonstrated that treatment with 12-hydroxy-eicosatetraenotic acid, a major metabolite of 12-LOX, increased MCP-1 expression in cardiac fibroblast and endothelial cells but not in cardiomyocytes. To determine the role of MCP-1 in the heart of 12LOX transgenic mice, we treated these mice with 7ND, an inhibitor of MCP-1, for 32 weeks. Chronic treatment with 7ND attenuated infiltration of macrophages into the myocardium and prevented systolic dysfunction and cardiac fibrosis in 12-LOX transgenic mice. Likewise, disruption of 12-LOX significantly reduced expression of MCP-1 and infiltration of macrophages in the heart, thereby inhibiting cardiac remodeling after myocardial infarction. Our in vitro and in vivo results suggest that cardiac 12-LOX is critically involved in the development of heart failure and that inhibition of 12-LOX will be a novel target for the treatment of this condition.
Abstract 3451: Risk for Coronary Artery Disease at the 9p21.3 Locus Is Abolished by a Protective Locus at 8p21.3 Identified by a Genome-Wide Association Study
Authors: Stewart Alexandre F , McPherson Ruth , Chen Li , Williams Kathryn , Doelle Heather , Davies Robbie , Dandona Sonny , Kavaslar Nihan , Ruthberg Julie , Ewart Gwen , LaRose Rosemary , Vo Lan , Wang Yanquing , Lau Paulina , Labinaz Marino , Chow Benjamin , Wells George A , Roberts Robert
Published: July 2021

Background
: Coronary artery disease (CAD) is the leading cause of death in the western world. The only common genetic risk locus identified to date by us and others in genome wide association studies (GWAS) resides at 9p21.3.

Methods:
The Ottawa Heart Genomic Study compared genotypes of 1,542 early onset CAD patients (average 49y) and 1,455 elderly control Caucasians (average 75y).

Results:
Here, we report two protective haplotypes in the 3′ region of the lipoprotein lipase (LPL) gene at 8p21.3. LPL promotes lipolysis of circulating triglycerides (TG) and increases levels of atheroprotective high density lipoprotein (HDL) cholesterol. Since the 8p21.3 haplotypes are protective, we tested their interaction with the 9p21.3 risk allele. The rs10503669 haplotype associated with elevated TG and HDL levels but did not affect risk from 9p21.3. In contrast, the rs17411031 haplotype was not associated with TG or HDL levels and showed a significant interaction that abolished 9p21.3 risk independent of known risk factors. The interaction was confirmed using data of the Wellcome Trust Case Control Consortium. Excluding carriers of the interacting allele, accuracy of 9p21.3 to predict CAD risk is dramatically improved.

Conclusion:
Identification of the first modifier gene of the 9p21.3 risk locus for CAD improves CAD risk assessment and provides novel insight into coronary atherosclerosis. This protective 8p21.3 locus may provide a target for novel therapy.
Abstract 3471: Ablation Of Phospholamban And Sarcolipin Has A Deleterious Effect On SERCA Pump Activity And Cardiac Contractility
Authors: Shanmugam Mayilvahanan , Gao Shumin , Kranias Evangelia , Periasamy Muthu , Babu Gopal J
Published: July 2021

Recent studies have been directed towards the potential therapeutic value of improving the sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA) function in the failing myocardium. Overexpression of SERCA pump or inhibiting the function of phospholamban (PLB) has been shown to improve the cardiac function in failing myocardium. Towards this goal, we enhanced the SERCA pump activity in both atria and ventricle by ablating its key regulators, PLB and sarcolipin (SLN). The homozygous double knockout (dKO) pups were delivered in Mendelian ratio and reached adulthood without any visible abnormalities. However, these mice develop cardiac hypertrophy. The heart weight to body weight ratio significantly increased in 3– 4 months old dKO mice (WT-3.08±0.11 vs. dKO-4.14±0.14) and is associated with enlargement of myocytes (WT-117±8 μm2 vs. dKO-166±10 μm2). Ablation of PLB and SLN did not affect the expression of major Ca2+ handling proteins including SERCA2a, calsequestrin, L-type Ca2+ channel and ryanodine receptor in both atria and ventricles. Echocardiographic measurements showed increased diastolic (WT-0.90±0.02 mm vs. dKO-1.26±0.07 mm) and systolic (WT-1.33±0.03 mm vs. dKO-1.68±0.08 mm) septal wall thicknesses and diastolic (WT-0.91±0.02 mm vs. dKO-1.15±0.07 mm) and systolic (WT-1.15±0.03 mm vs. dKO-1.35±0.07 mm) post wall thickness in 3– 4 months old dKO mice hearts. However, left ventricular end-diastolic dimension (LVEDD), LV end-systolic dimension and ejection fraction (EF, %: WT-0.73±0.12 vs. dKO-0.69±0.03) are not very different from wildtype indicating preserved contractile function in the dKO mice. Our studies therefore suggest that the enhanced Ca2+ sensitivity of cardiac SERCA pump can progressively lead to cardiac hypertrophy and may have a deleterious effect on cardiac function during pathological conditions.
Abstract 3473: Down Regulation of Class I Histone Deacetylase in the Deficit of Cardiac Lineage Protein-1 Protects Heart from Ischemia Reperfusion Injury
Authors: Gurusamy Narasimman , Lekli Istvan , Mascareno Eduardo , Siddiqui M.A.Q. , Das Dipak K
Published: July 2021

Cardiac lineage protein-1 (CLP-1), the mouse homologue of human Hexim1 protein, exerts inhibitory control over transcriptional elongation factor-b (P-TEFb) of RNA transcript elongation, and this inhibitory control of CLP-1 over P-TEFb is released under hypertrophic stimuli. Histone deacetylases (HDACs) are known to repress the transcription of genes via maintaining compact chromatin structure. Reports have shown that class II HDACs repress the growth of myocytes, whereas, class I HDACs promotes cellular growth. A recent report suggests that inhibition of HDACs can protect the heart from ischemic injury. In the present study, we determined the role of HDACs under the down regulation of CLP-1 in ischemia reperfusion (I/R) injury and in the regulation of cardiac hypertrophy. Our results showed that the nuclear expression of class I HDACs such as HDAC1, HADC2 and HDAC3 were reduced in CLP +/− mice compared to CLP +/+ mice. In contrast to class I HDACs, class II HDACs such as HDAC4, HDAC5 and HDAC7 were not significantly altered between CLP +/− and CLP +/+ mice. When the isolated mice hearts were subjected to 30 min of ischemia followed by 120 min of reperfusion, cardiac functional parameters like aortic flow, left ventricular developed pressure and left ventricular dp/dt were significantly improved in CLP +/− mice compared to CLP +/+ mice. The percentage of myocardial infarct size after I/R injury was significantly less in CLP +/− mice than CLP +/+ mice. The occurrence of cardiac hypertrophy in CLP +/− mice was confirmed by the increased expression of fetal gene expression markers such as β-myosin heavy chain, atrial natriuretic factor and α-skeletal actin. The nuclear expression of HDAC1, HDAC2 and HDAC3 were reduced after I/R injury in CLP +/− mice compared to control CLP +/− mice. However, HDAC4, HDAC5 and HDAC7 were not altered after I/R injury in CLP +/− mice. In conclusion, our results suggest that the down regulation of class I HDACs but not class II HDACs in CLP-1 +/−mice could cause cardioprotection against I/R injury, and may also responsible for the elicitation of cardiac hypertrophy.
Abstract 3484: High Plasma Levels of Endogenous Free Dopamine Predict an Adverse Outcome in Patients with Heart Disease and Chronic Kidney Disease
Authors: Sano Keita , Kodama Yasushi , Hirano Mitsumasa , Takishima Isao , Makino Aritaka , Nakamura Takamitsu , Kitta Yoshinobu , Kawabata Kennichi , Obata Junei , Kugiyama Kiyotaka
Published: July 2021

Sympathetic activity is heightened in chronic kidney disease (CKD). The increased sympathetic activity is a risk of future cardiovascular disease (CVD) events. Dopamine, a precursor of norepinephrine biosynthesis, is metabolized in the kidney and excreted into urine, and its plasma levels are increased in renal dysfunction. Thus, we examined whether plasma levels of endogenous dopamine may be related to future CVD events in patients with CKD. A total of 840 patients in stable condition with chronic heart disease (622 patients with ischemic heart disease and 218 with non-ischemic heart disease) (586 patients with GFR ≥ 60 ml/min/1.73 m
2
[normal/mild CKD]; 153 patients with 60 > GFR ≥ 30 [moderate CKD]; 101 patients with 30 > GRF [advanced CKD]) were prospectively followed-up for 5 years or until one of the following CVD events: cardiac death, non-fatal myocardial infarction, unstable angina pectoris requiring unplanned revascularization, worsening heart failure requiring hospitalization, or stroke. Plasma levels of endogenous free dopamine at entry were measured by high-performance liquid chromatography. Patients with a higher stage of CKD had higher dopamine levels than patients with a lower stage (63.0 ± 5.5, 45.0 ± 5.1, and 26 ± 1.2 pg/mL, respectively, ρ = 0.54, p < 0.0001 Spearman rank correlation test). During the follow-up period, 233 patients had CVD events (cardiac death in 54, myocardial infarction in 18, others events in 161) (113 events [19%] in normal/mild CKD, 64 [42%] in moderate CKD, and 56 [55%] in advanced CKD). In patients with moderate CKD and advanced CKD, higher dopamine levels (≥ 40 pg/mL, defined by ROC analysis) were the strongest predictor of CVD events (HR 2.1 and 3.3, 95% CI 1.2 – 3.7 and 1.5 – 7.2, p = 0.006 and 0.003, respectively) in a multivariate Cox hazards analysis using plasma norepinephrine and BNP levels, hypertension, and hyperlipidemia as covariates. In contrast, dopamine levels did not have predictive value in normal/mild CKD (HR 1.2, 95% CI 0.7 – 2.0, p = 0.5). High plasma levels of endogenous free dopamine are a risk factor for future CVD in patients with CKD independently of norepinephrine levels. Increases in plasma endogenous free dopamine levels may serve as a mechanistic link between CKD and CVD.
Abstract 3546: Heart Failure-Derived Macrophages Induce Myocyte Oxidative Stress and Contractile Dysfunction in a Juxtacrine and iNOS-Dependent Manner
Authors: Kingery Justin R , Lewis Robert K , Partridge Charles R , Ismahil Ameen , Cardwell Ernest , Hamid Tariq , Jones Steven P , Prabhu Sumanth D
Published: July 2021

The failing heart exhibits low-level, but progressive, tissue macrophage infiltration. Although macrophages are sources of pro-oxidants, whether macrophage-myocyte interactions are of consequence in heart failure (HF) is unknown. We
hypothesized
that macrophage-myocyte juxtacrine interactions can induce myocyte dysfunction in HF. Wild-type (WT, C57BL/6) mice underwent coronary ligation (HF group) or sham operation. At 4 weeks in the HF group, echocardiography and hemodynamics confirmed LV dysfunction and remodeling. Flow cytometry showed a larger number of activated blood monocytes (F4/80+CD11b+: 11±2% vs. sham 7±2%, p <0.05), and cardiac MOMA-2 immunostaining revealed a 2-fold increase in tissue activated macrophages. To assess macrophage-myocyte interactions, thioglycollate-elicited peritoneal macrophages were collected from WT naíve and HF mice, macrophages were co-cultured with isolated adult WT myocytes, and myocyte contraction was assessed in the presence or absence of myocyte-macrophage physical contact. Also, as macrophages lacking inducible nitric oxide synthase (iNOS) exhibit blunted activation, to deconstruct the role of macrophage activation, peritoneal macrophages were collected from ligated iNOS−/− HF mice. In the absence of macrophage association, myocyte contraction (1 Hz) was 9.5±1.3%. Contraction was significantly (p < 0.05) depressed upon physical contact with either WT HF macrophages (4.6 ± 1.0%) or LPS-stimulated naíve WT macrophages, but not upon contact with unstimulated naíve WT macrophages. In contrast, physical contact with iNOS KO HF macrophages did not depress contraction (shortening 8.9±1%, p = NS), even with LPS pre-stimulation. Myocytes were loaded with the fluorescent indicators DCF-DA or HKGreen-2 to monitor reactive oxygen species (ROS) and peroxynitrite, respectively. Myocyte attachment to WT HF macrophages induced a 4-fold increase in myocyte ROS and peroxynitrite over baseline, events that did not occur with attachment to iNOS KO HF macrophages. We conclude that direct physical interaction with activated macrophages in HF is sufficient to induce myocyte oxidative and nitrative stress and contractile dysfunction, and this is dependent, at least in part, on macrophage-derived iNOS.

This research has received full or partial funding support from the American Heart Association, AHA Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).
Abstract 3558: Time of Day: A Vital Determinant in Diet-Induced Cardiometabolic Syndrome Development
Authors: Bray Molly S , Tsai Ju-Yun , Boland Brandon , Kueht Michael , Egbejimi Oluwaseun , Blasier Zachary , Kelly Rebecca , Zoller Jason , Young Martin E
Published: July 2021

Excess caloric intake is strongly associated with the development of obesity, type 2 diabetes mellitus, and cardiovascular disease (CVD). Research efforts have focused attention primarily on the quality (i.e., nutritional content) and/or quantity of calories as potential causes for diet-induced pathology. Little is known regarding how the
timing
of nutrient ingestion influences CVD development, despite growing acceptance that biological rhythms strongly affect cardiovascular physiology and pathophysiology. We therefore hypothesized that the time of day at which dietary fat is consumed profoundly influences the development of the cardiometabolic syndrome. Mice were initially fed either control or high fat diets (10% and 45% calories from fat, respectively) in an
ad libitum
fashion. As expected, 16 weeks ad libitum high fat feeding resulted in increased body weight gain (13%), increased percent body fat (23%), increased ventricular weight (11%), and myocardial contractile dysfunction (33% decrease in cardiac power, as assessed in
ex vivo
working mouse heart perfusions), relative to control mice (p<0.05). A separate set of mice were next fed only during the 12hr active/awake/dark phase, and were divided into early fat (EF) and late fat (LF) groups (access to high fat diet only during the first 4 hours versus the last 4 hours of the active phase, respectively; mice were allowed access to control diet for remaining active phase hours). LF feeding resulted in increased body weight gain, increased percent body fat, decreased glucose tolerance, decreased skeletal muscle oxidative capacity, induction of myocardial
anf
expression, and decreased cardiac power, relative to EF feeding, despite virtually identical quantity and quality of calories consumed (see Table

). These data reveal that the consumption of dietary lipids during the end of the active phase (i.e., dinner for humans) significantly contributes to diet-induced cardiometabolic syndrome development.