bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 704: Profiling Differential Response to Myocardial Infarction by Single Cell Analysis of the Cardiac Interstitium
Authors: Forte Elvira
Published: October 2019

Myocardial infarction (MI) is one of the main causes of mortality in Western Countries. The non-regenerative adult mammalian heart responds to injury with the formation of a scar. The outcome of repair is highly dependent on interstitial cells and on the delicate interplay between the inflammatory response and fibrosis, which leads to adverse ventricular remodeling and heart failure or cardiac rupture in the early phase post-injury. Our data show that inbred mice strains have different reliance to MI, suggesting differences in the cellular response to the injury. To profile the dynamic response of cardiac interstitial cells to MI and their embryological origin, we performed unbiased scRNAseq analysis of cardiac interstitial cells at homeostasis and 1, 3, 5, 7, 14, 28 days post-injury from transgenic mice on C57bl/6j background (B6), expressing ZsGreen under the control of the epicardial marker Wt1 (

Wt1
Cre
;RosaZsgreen
f/+
)

. About 38,600 cells were captured using the 10xChromium technology and we distinguished 16 main cell populations that evolved over time. By high resolution sub-clustering of stromal cells, 7 main subpopulations were identified. Of these, one was endocardial derived and present at all time points, the others were epicardial derived (ZsGreen+) and included 3 populations of fibroblasts present at different frequencies at all time points, a transient population of immune-response activating fibroblasts (IR) prevailing at day1 and preceding the appearance of myofibroblasts between day 3 and 7 and matrifibrocytes prevalent at day 14 and day 28. To determine the role of cell composition in different reparative outcomes, we compared the data on the C57BL/6J background with 129S1/SvlmJ (129), the inbred strain presenting the highest frequency of cardiac rupture (70%) among the 9 tested. We observed increased number of activated IR and myofibroblasts in 129 3 days post-MI. 129 also showed higher level of collagens, chemokines, fibrin deposition and acute phase response genes. The hyperactivation of myofibroblasts was possibly mediated by the enhanced responsiveness to the Renin-Angiotensin System, currently one of the main targets of clinical therapies.
Abstract 720: Prenatal Exposure of Cigarette Smoke Impacts Cardiac Regeneration
Authors: Schimmel Katharina , Morgado Isabel , Tsai Cheng-Yu , Evangelisti Alessandro , Fisch Sudeshna , Ngoy Soeun , Lee Dian-Jian , Dangwal Seema , Alexander Kevin , Ward Jennifer E , Liao Ronglih
Published: October 2019

Background:
Neonatal mammalian hearts have the unique characteristic to fully repair and regenerate following injury. This discovery has led to visionary endeavors to understand and subsequently reawaken regeneration processes in the human adult heart that are lost after birth, contributing to heart failure and death after ischemic insults.
In utero
exposure to tobacco smoke has detrimental effects on fetal development and growth. However, the consequences of prenatal exposure to cigarette smoke on murine neonate’s cardiac regeneration have never been explored.

Methods:
To study the impact of cigarette smoke during the entire pregnancy on cardiac regeneration in the offspring, plugged female wild type C57LB/6J mice were exposed either to cigarette smoke (n=6) or filtered air (control, n=6). Two days after birth, 5 pups from each litter were assigned to the two experimental groups: Myocardial infarction (MI) and sham. Detrimental effects of
in utero
exposure to tobacco smoke on the recovery of cardiac function following MI surgery were followed using two-dimensional speckle tracking echocardiography and strain imaging. To investigate the underlying mechanism, cardiac tissue of the infarct and remote zone was collected for non-coding RNA analysis.

Results:
In utero
exposure to cigarette smoke significantly compromised cardiac regeneration in neonates. At both early and late time points in the phase of cardiac repair, hearts of neonates exposed to cigarette smoke during pregnancy showed a marked impairment of cardiac regenerative potential. In particular, ejection fraction, fractional area change and shortening, as well as left ventricular internal diameter during systole remained pathologically changed following MI insult in the smoked group until the very endpoint.

Conclusions:
Collectively, we here provide evidence that
in utero
exposure to cigarette smoke strongly compromises cardiac regeneration in the newborn offspring. This result reinforces smoking cessation during pregnancy. Additionally, understanding the changes in non-coding RNA expression in a setting of preserved versus disrupted repair of the heart might be an important first step towards the identification of key cellular processes in cardiac regeneration after injury.
Abstract 738: Comparative Cardiotoxicity of Tyrosine Kinase Inhibitors Ponatinib and PF114
Authors: Singh Anand P , Umbarkar Prachi , Glennon Michael S , Zhang Qinkun , Chilov Ghermes , Becker Jason R , Lal Hind
Published: October 2019

Background:
The advent of tyrosine kinase inhibitor (TKI) targeted therapy revolutionized the treatment of chronic myelogenous leukemia (CML) patients, leading to a significant impact on remission rates and overall survival. However, cardiotoxicity associated with these targeted therapies put the cancer survivors at greater risk. Ponatinib is an example shown unexpected wide spectrum of cardiotoxic complications which limits its clinical applications. A newly designed 4
th
-generation tyrosine kinase inhibitor PF114 having better target spectra in CML, could be a potential alternative to ponatinib. But the cardiotoxic potential of PF114 is not yet fully understood.

Objectives:
The objectives of the present study were to compare cardiotoxicity of ponatinib with PF114.

Methods:
In this study, we employed zebrafish transgenic BNP reporter line that expresses luciferase under control of the nppb promoter (
nppb
: F-Luciferase) to compare cardiotoxic potential of TKIs. We also exploited NRVMs to explore cardiotoxic mechanism associated with ponatinib and PF114.

Results:
We observed that increasing dose of PF114 showed minimal induction of the BNP reporter compared to ponatinib. PF114 did not reduce the ventricular fractional shortening in zebrafish as compared to ponatinib. Consistently, in cultured rat cardiomyocytes, PF114 could not reiterate the same cardiotoxic effect of ponatinib as depicted by cell viability and TUNEL assay. Mechanistically, PF114 has shown no effects on essential prosurvival AKT and ERK signaling pathway while ponatinib is known to exerts its cardiotoxic effects by their inhibition. Additionally, the allosteric tyrosine kinase inhibitor ABL001(Asciminib) in combination with PF114 depicts less pronounced cardiotoxic effects than ponatinib in similar settings.

Conclusions:
This study provides further rationale to utilize zebrafish for the prediction of cardiotoxicity of anticancer drugs. The comparative cardiotoxicity of ponatinib and PF114 suggest that PF114 is a significantly less cardiotoxic analogue and safer treatment option than ponatinib for CML patients harboring BCR/ABL-T315I “gatekeeper” mutation.
Abstract 746: The Effect of Aging-related Metabolic Disorder on Resistin Expression and Valvular Heart Disorder
Authors: Bouchareb Rihab , Saadallah Najla , Hajjar Roger , Lebeche Djamel
Published: October 2019

Calcific aortic valve stenosis (AS) is the most prevalent valvular heart disease and the third most frequent cardiovascular disease after coronary artery disease and hypertension. AS is characterized by a period of asymptomatic progressive valve calcification, which eventually leads to the development of a severe stenosis and to the onset of symptoms. Visceral obesity is a strong predictor of type-2 diabetes and is associated with insulin resistance. In addition, increased visceral adiposity leads to an increase in systemic release in resistin (Retn) and possibly interleukins. Elevation of circulating adipokines such as Retn plays a role in the development of muscle insulin resistance. Moreover, higher Retn levels were associated with increased valvular inflammation and calcification in the elderly patients, whereas this association was not observed among middle-aged patients. In the current study, we explored the potential role of Retn in ectopic valvular calcification. Analyses of scanning electron microscopy micrographs of calcified aortic valve showed that Retn is more abundant next to calcified nodes. These data corroborate with increased levels of circulating Retn in apoE mice that were fed a high fat high sucrose diet for 24 weeks. Moreover, apoE-/-Retn-/- double KO mutant mice have lower plasma cholesterol level compared to apoE KO mice. In addition, knocking down Retn decreased valve lipid infiltration, lowered glycaemia, calcium nodes and reduced fibrosis. In conclusion, Retn may be a new pharmacological target to stop the progression of aortic stenosis in patients with visceral obesity.
Abstract 751: The Inhibition of PARP Provides Greatercardioprotection in Mice With Heart Failure
Authors: Katoh Manami , Nomura Seitaro , Ko Toshiyuki , Fujita Kanna , Yamada Shintaro , Ito Masamichi , Aburatani Hiroyuki , Komuro Issei
Published: October 2019

The most cause of death in Japan is cancer and the second is heart disease. It is expected that the number of patients with both diseases will continue to increase in the increasingly aging society. Cardiotoxicity by anticancer agents has been recognized for a long time, for example alkylating agents, antimetabolites, proteasome inhibitors, and anthracycline anticancer agents. As cancer patients and heart failure patients increase, the number of patients suffering both diseases at the same time will naturally increase. In recent years, many new anticancer drugs have been developed and are on the market in succession, and the research on cardiotoxicity in each drug has not kept up. In 2017, from our group, there was a report that inflammation was caused by an increase in cardiomyocyte single strand breaks in pressure-overload heart failure model mice, and that inflammation contributes to declining cardiac function . DNA repair related protein PARP1 plays an important role in the repair mechanism of SSBs. PARP inhibitor disturbs its mechanism and exerts antitumor effect by causing tumor cells to become apoptosis. Therefore, we hypothesize that orally administered PARP inhibitors in heart failure patients will inhibit DNA repair by PARP, thus exacerbate inflammation and impair cardiac function more. To examine this hypothesis, we administered a representative PARP inhibitor, olaparib to pressure-overload heart failure model mice. Surprisingly, the cardiac function was improved in the olaparib administrated group. PARP is known to recruit DNA repair related proteins through synthesis of PAR (Poly-ADP-Ribose) on the DNA damage sites. In recent reports, several studies have shown that the accumulation of PAR would be involved in the worsening of the disease state in cerebellar degenerative ataxia and Parkinson's disease. In our immunostaining study of PAR using the heart tissue specimens from 58 patients with dilated cardiomyopathy, PAR was stained much more in patients with poor outcomes. I believe that PARP inhibitors may have a cardioprotective effect by preventing the accumulation of PAR, and we are now conducting further analyzes.
Abstract 755: Fas Ligand- and Nitric Oxide-Releasing Stent
Authors: Kural Mehmet H , Wang Juan , Gui Liqiong , Li Guangxin , Yuan Yifan , Leiby Katherine L , Quijano Elias , Tellides Geroge , Saltzman W. Mark , Niklason Laura E
Published: October 2019

To restore blood flow in stenotic vessels such as coronary arteries, metallic vascular stents are often used. However, stent deployment under high pressure leads to vascular wall and smooth muscle (SMC) damage, with compensatory thrombosis, inflammation, and neointimal proliferation of SMCs. These events result in vessel re-occlusion and treatment failure. In an effort to prevent intimal hyperplasia and treatment failure, drug-eluting stents were developed roughly 15 years ago. These stents release global inhibitors of proliferation (eg. “mTOR inhibitors”) which stop the growth of SMC as well as vascular endothelial cells (EC). Despite being widely used, drug-eluting stents mandate long-term treatment with potent anticoagulants. This is because mTOR inhibitors prevent re-endothelialization of the stent surface, leaving the clot-inducing metal exposed to the bloodstream. Yet, long-term dual antiplatelet therapy leads to increased risk of bleeding/stroke and myocardial infarction. Here, we leveraged the fact that nitric oxide (NO) increases Fas receptors on the SMC surface. Fas forms a death-inducing complex upon binding to Fas ligand (FasL), while ECs are relatively resistant to this pathway (Figure 1). Selected doses of FasL and NO donor synergistically increased SMC apoptosis and inhibited SMC growth more potently than did everolimus or sirolimus, while having no significant effect on EC viability and proliferation. We verified this differential effect in an ex vivo pig coronary artery model, where the intimal thickening was inhibited by the drug combination, but endothelial viability was retained. We also deployed FasL and NO donor-releasing ethylene-vinyl acetate copolymer (EVAc)-coated stents into pig coronary arteries, and cultured them in perfusion bioreactors for one week. FasL and NO donor, released from the stent coating, killed SMCs in the proximity of the stent struts, even in the presence of flow rates mimicking those of native arteries. Therefore, the FasL-NO donor-combination has the potential to prevent intimal hyperplasia and in-stent restenosis, without harming endothelial restoration, and hence may be a superior drug delivery strategy for drug-eluting stents.
Abstract 765: Sex Difference in Cardiomyocyte Functional Etiology in Heart Failure With Preserved Ejection Fraction
Authors: Curl Claire L , Raaijmakers Antonia J , Bell James R , Harding Tristan W , Harrap Stephen B , Delbridge Lea M
Published: October 2019

Heart failure with preserved ejection fraction (HFpEF) accounts for >50% of heart failure patients and is particularly prevalent in women. A mechanistic understanding of the intrinsic cardiomyocyte pathology in HFpEF is limited with no selective treatments identified. Gender-specific aspects of HFpEF etiology have not been well characterized. The aim of this study was to experimentally evaluate sex difference in cardiac and cardiomyocyte performance in HFpEF, using a novel recently developed rodent model of HFpEF - the ‘Hypertrophic Heart Rat’.

Echocardiography was performed in 50 week male and female HHR/NHR (Normal Heart Rat) using a GE Vivid 9. Fibrosis measurements employed picrosirius red staining. Single myocyte force, shortening and intracellular Ca
2+
measurements were obtained (Myostretcher, Ionoptix).

Male and female HHR hearts were hypertrophic (M: 4.9±0.3 vs 3.7±0.1; F: 6.6±0.5 vs 4.4±0.1mg/g CWI) with significant diastolic dysfunction (M:27.8±1.4 vs 18.0±1.7; F: 28.9±1.2 vs 18.9±1.0 E/E’) and preserved systolic function (M:78.4±1.9 vs 74.5±1.3; F: 80.9±1.3 vs 78.1±0.8 EF). Female HHR, but not males, exhibited diffuse interstitial fibrosis (M: 3.33±0.1 vs 3.51±0.1; F: 11.45±0.4 vs 8.44±0.2%). In male, but not female, cardiomyocytes, hypercontractility and high cytosolic operational Ca
2+
levels were observed (64% and 116% increase respectively). The force-length gradient produced by serial stretches of isolated cardiomyocytes was increased in male and female HHR (M:6.8±0.4 vs 4.3±0.8; F: 7.4±0.5 vs 3.0±0.4 nN/%SL stretch) indicating stiffer cardiomyocytes in the HFpEF hearts. The increase in stiffness was significantly accentuated in females.

This study indicates that in HFpEF the cardiomyocyte functional etiology is different in males and females. Whilst in males the relationship between cardiomyocyte hypercontractility is the most prominent characteristic of HFpEF, in females the remodelling of the extracellular matrix exacerbates instrinsic myocyte stiffness. Further investigation of the molecular pathways involved in these processes is required for identification of potential therapeutic targets.
Abstract 770: Potential Explanation of the Mechanism for Loss of Function with G233D Mutation in Platelet Glycoprotein Ibα: Results fromMolecular Dynamics Simulation
Authors: Goto Shinichi , Oka Hideki , Ayabe Kengo , Ybushita Hiroto , Hasebe Terumitsu , Yokota Hideo , Takagi Shu , Sano Motoaki , Goto Shinya
Published: October 2019

Background:
Platelet play crucial role for the onset of acute coronary syndrome. The initial binding following vessel injury is exclusively mediated by the binding of von Willebrand factor (VWF) and platelet glycoprotein (GP) Ib
α
. Previous biological experiments revealed loss of function of platelet expressing GPIb
α
with G233D mutation.

Objective:
To elucidate the mechanism underlining the loss of function in G233D mutant.

Methods:
Dynamic fluctuating three-dimensional structures and the Potential of Mean Force (PMF) were calculated for the binding of VWF and platelet GPIb
α
in wild-type or G233D mutant. PMF were calculated at each 0.5Å for 25 Å to 65 Å of mass center distance between GPIb
α
and VWF. The energy required to dissociate the bond between GPIb
α
and VWF was calculated by subtracting the lowest PMF from the PMF at 65 Å mass center distance. Chemistry at HARvard Molecular Mechanics (CHARMM) force field with NAnoscale Molecular Dynamics (NAMD) was used for calculation. The initial structure of each mutant was obtained by inducing single amino-acid substitution with Visual Molecular Dynamics (VMD) to the stable water-soluble binding structure of wild-type VWF and GPIb
α
.

Results:
The energetically most stable binding structure of VWF and GPIb
α
in wild-type and G233D did not differ substantially (Figure panel A). However, The energy required to dissociate the bond between GPIb
α
and VWF was 4.32 kcal/mol (19.5 %) lower for G233D mutant compared to wild-type (Figure panel B).

Conclusions:
Our results suggest that the reduction of dissociation energy of single molecule of G233D GPIb
α
mutant as a possible explanation for the reduced platelet adhesion under blood flow condition.
Abstract 771: Identification of Pathological Pathways in Hypertrophic Cardiomyopathy via Single Nuclei RNA-Sequencing
Authors: Larson Amy , Bowditch Heather K , Rastegar Hassan , Chen Frederick , Rowin Ethan , Maron Marty , Huggins Gordon , Chin Michael T
Published: October 2019

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiovascular disease, is characterized by left ventricular hypertrophy, is often asymmetric, and is unexplained by secondary causes. The molecular and cellular basis of this asymmetric hypertrophy is unknown. HCM has been thought of as a disease of the sarcomere, with mutations in more than a dozen sarcomere-associated proteins correlated with HCM. These mutations, however, exhibit large phenotypic heterogeneity with little correlation between mutation and severity. Treatment of HCM is limited and thus, understanding what drives the disease at the molecular and cellular level could lead to novel treatment strategies.

Obstructive HCM is a severe form of the disease where the left ventricular outflow tract is obstructed. Patients with obstructive HCM often undergo septal myectomy surgery to remove the obstructive lesion. To understand the underlying mechanisms of obstructive HCM, we performed single nuclei RNA-sequencing on thousands of nuclei from 11 patients’ myectomy samples to determine the gene expression patterns in individual cells and identify the cell populations in those lesions. We used rejected donor hearts that do not exhibit any LV hypertrophy or have any major cardiovascular defects as control samples to compare the gene expression patterns in those cardiomyocytes to those in obstructive HCM lesions.

We performed principal component analysis and hierarchical clustering on the RNA expression counts to identify the different cell populations in each dataset, and looked for cell populations that were present across all HCM datasets. This analysis revealed distinct cardiomyocyte populations in obstructive lesions. Differential gene expression analysis among these clusters revealed a minority cardiomyocyte population in the obstructive lesions that exhibited a relatively normal gene expression pattern, while the majority cardiomyocyte cluster exhibited a shift in gene expression to genes involved in growth pathways. This abnormal cardiomyocyte population was absent in the control datasets. These findings imply that obstructive lesions seen in HCM may arise from abnormal growth of a subset of cardiomyocytes that could possibly be targeted by novel therapeutics.
Abstract 794: Endothelial Specific Ablation of Estrogen Receptor Alpha Rapid Signaling Revealed Exacerbated Vascular Remodeling Response
Authors: Liu Pangyen , Hiroi Yukio , Ueda Kazutaka , Fukuma Nobuaki , Li Yuxin , Noma Kensuke , Liao James K.
Published: October 2019

Background:
Estrogen exerts complex physiological effects via its rapid (non-genomic) and genomic actions. In particular, rapid signaling of estrogen receptor alpha (ERα) has been implicated in the vasculo-protective effects, in which both endothelial and smooth muscle cells might be involved. However, no prior studies have determined the role of ERα rapid signaling in the endothelium. This study aims to clarify the impact of ERα rapid signaling in the vasculo-protection, using a novel mouse model lacking endotheilal-specific ERα rapid signaling.

Methods and Results:
In immunoblotting, p85α and pGSK3β, non-genomic pathway downstream signals, were reduced in ERα mutants RR259/260AA with estradiol (E2) stimulation at concentrations of 10
-11
and 10
-10
M . ERE-luciferase assay demonstrated E2 induced genomic pathway activity was preserved even in ERα mutants RR259/260AA. We generated a mouse model in which rapid signaling of ERα was ablated in the endothelium by crossing Tie2-Cre transgenic mice with floxed ERα mutants (RR 259/260 AA) in which p85α and ERα interaction was disrupted. In endothelial cells isolated from ERα
KI/KI
Tie2
cre/+
animals, E2 at a concentration of 10
-8
M failed to induce phosphorylation of Akt, confirming the absence of ERα rapid signaling. Baseline characteristics at 8 to 12 weeks of age were undistinguishable between the genotypes, including body weight (KI:19.2±0.366 g vs WT: 18.9±0.356 g ) systolic blood pressure (KI:106.1±3.336 vs WT: 103.5±2.133 mmHg) and echocardiographic fractional shortening (KI:54.4±0.751 vs WT: 55.19±0.947 %). We then assessed how vascular remodeling process was impacted in a carotid artery wire injury model. Histological analyses with Elastica van Gieson staining two weeks after induction of injury revealed that wall thickness and area of medial layer composed mainly of smooth muscle cells were significantly increased in ERα
KI/KI
Tie2
cre/+
mouse, as compared to wild types (KI:44.85±1.948 μm vs WT: 29.32±1.753 μm , KI:42,047±1,560 μm
2
vs WT: 32,815±1,211 μm
2
, respectively ).

Conclusions:
Our results demonstrate that the rapid signaling of ERα in the endothelium critically regulates vascular smooth muscle cell growth after vascular injury, suggesting the essential role to vascular remodeling.
Abstract 804: Elimination of Cells Expressing Senescence Associated Glycoprotein Attenuates Theatherosclerotic Diseases
Authors: Suda Masayoshi , Shimizu Ippei , Yoshida Yohko , Katsuumi Goro , Hayashi Yuka , Ikegami Ryutaro , Nagasawa Ayako , Nakao Masaaki , Fruuchi Ryo , Ozawa Takuya , Minamino Tohru
Published: October 2019

Although accumulation of senescent vascular endothelial cells impairs the vessel homeostasis and promote atherosclerotic diseases, underlying mechanisms are largely unknown. We found that Senescence associated glycoprotein (SAGP) expression was significantly increased in human endothelial cells undergoing replicative senescence. SAGP expression in aorta was significantly increased in Apo-E knockout mice. Furthermore, mean SAGP expression was significantly higher in the leukocytes of the patients with atherosclerotic diseases.These data suggest that SAGP would become the novel cellular senescence and/or atherosclerotic disease marker. The genetic deletion of SAGP resulted in high level of mitochondrial reactive oxygen species (ROS) and promoted premature senescence in human endothelial cells. And this associated with suppression of mitochondrial autophagy, mitophagy, suggesting that SAGP protect cells from ROS by inducing mitophagy. Thus, wegenerated ApoE-KO / SAGP gain-of-function transgenic mice and found that atherosclerotic plaque burden was attenuated in these transgenic mice. In contrast, in SAGP / ApoE double knockout mice atherosclerosis were impaired. These data suggest that modulation of SAGPwould become a new therapeutic target for atherosclerotic diseases.Recently, it is reported that elimination of senescent cells (senolysis) reversibly improved pathological aging phenotypes. We generated SAGP-DTR (diphtheria toxin receptor) transgenic mice,inducible elimination of SAGP positive senescent cells upon administration of diphtheria toxin. Elimination of SAGP expressing cells significantly reduced the atherosclerotic plaque burden. Furthermore, we developed a cytotoxic vaccine targeting SAGP. Administration of SAGP vaccine to ApoE-KO mice significantly reduced atherogenesis. These data indicate that targeting SAGP-positive cells could be a strategy for senolytic therapy.
Abstract 806: PD-1 in Cardiac Immune Responses After Ischemic Injury or Transverse Aortic Constriction
Authors: Zlotoff Daniel A , Xiao Chun Yang , Grange Robert M , Iwamoto Yoshiko , Freeman Gordon , Rosenzweig Anthony
Published: October 2019

Background:
Heart failure is associated with inflammation, but the precise immune mechanisms that contribute to cardiomyopathy remain elusive. Myocarditis is a rare toxicity of checkpoint inhibitors, which are oncologic therapies that block signaling through the immunoinhibitory molecule PD-1. We investigated whether PD-1 attenuates cardiac immune responses.

Methods:
Expression of PD-1 and its ligand PD-L1 on cardiac cell populations were assessed by flow cytometry. The functional implications of PD-1 signaling were determined using PD-1 blocking antibodies (Abs) in two surgical models of cardiac injury: ischemia/reperfusion and transverse aortic constriction (TAC). Ischemic injury was measured by troponin release and histology. Systolic function was assayed by echocardiography, diastolic function by invasive pressure-volume loops, and fibrosis by histology.

Results:
PD-L1 is expressed on most cardiac endothelial cells and a small fraction of cardiomyocytes. While intracardiac T cells do not express PD-1 under baseline conditions, 15% of both CD4 and CD8 T cells express PD-1 after TAC. Blocking PD-1 signaling after ischemic injury did not affect troponin levels (5.8 ng/ml in mice receiving isotype control (IC) Ab vs 7.8 ng/ml in mice receiving anti-PD-1 Ab, p=0.26), size of infarct (1.6% of ventricular cross-sectional area for IC Ab vs 2.0% for anti-PD-1 Ab, p=0.27), or systolic function (fractional shortening 52.6% for IC Ab vs 53.2% for anti-PD-1 Ab, p=0.92). Similarly, abrogation of PD-1 signaling after TAC did not worsen systolic dysfunction (fractional shortening 36.8% for IC Ab vs 32.4% for anti-PD-1 Ab, p=0.38), diastolic function (dP/dT minimum -9445 mmHg/s vs -10950 mmHg/s, p=0.44; Tau 6.8 ms for IC Ab vs 6.0 ms for anti-PD-1 Ab, p=0.45), or the extent of cardiac fibrosis (1.4% of ventricular cross-sectional area for IC vs 1.7% for anti-PD-1 Ab, p=0.41).

Conclusion:
Collectively, these data do not implicate PD-1 as an important regulator of intracardiac immune responses in the injury models tested. This suggests that additional pathways may contribute to myocarditis occurring in cancer patients treated with anti-PD-1 Ab, the identification of which could provide insight into immune mechanisms active in heart failure.
Abstract 811: The Role of Mitochondrial Stress and the Late Sodium Current in Ibrutinib-Mediated Atrial Fibrillation
Authors: Menon Ambili , Chalzan Brandon , Hong Liang , sridhar Arvind , Zhang Meihong , Darbar Dawood
Published: October 2019

Background:
Ibrutinib (a Bruton’s tyrosine kinase inhibitor, BTKI) is rapidly becoming the treatment of choice for many B-cell malignancies, but its proarrhythmic effects may limit its widespread use. GWAS have consistently identified an AF risk locus on chromosome (chr) 4q25, and the nearest gene
Pitx2c
has been implicated in AF development as evidenced by increased AF in

Pitx2c
+/-

mice which serve as an experimental model for clinical AF.

Objective:
To assess the role of the late cardiac Na current (I
Na,L
) and oxidative stress in mediating increased susceptibility to AF in

Pitx2c
+/-

mice which are genetically prone to developing AF as well as in atrial human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) exposed to ibrutinib.

Method:

Pitx2c
+/-

mice were administered ibrutinib (30mg/kg/day IP) for 14 days while atrial hiPSC-CMs were exposed to ibrutinib for 48 hrs.

Results:
There was a graded increase in AF episodes and burden with the number of days the

Pitx2c
+/-

mice were exposed to ibrutinib versus controls (
Fig. 1A
). The action potential duration at 90% repolarization and the I
Na-L
were markedly prolonged (
Fig. 1B-C
). Furthermore, we observed a significant increase in mitochondrial fragmentation (
Fig. 1D-E
) and superoxide production (
Fig. 1F-G
) in ibrutinib treated atrial hiPSC-CMs versus controls.

Conclusion:
We showed in

Pitx2c
+/-

mice and atrial hiPSC-CMs that ibrutinib-mediated AF may in part be related to enhanced I
Na-L
and mitochondrial dysfunction leading to increased oxidative stress. Patients undergoing ibrutinib treatment may benefit from novel therapies which target mitochondrial dysfunction or the late cardiac sodium current.
Abstract 812: Mechanisms of Sinus Node Dysfunction and Chronotropic Incompetence in Rats with Heart Failure and Preserved Ejection Fraction
Authors: Mesquita Thassio , Cho Jae H , Zhang Rui , Goldhaber Joshua I , Marbán Eduardo , Cingolani Eugenio
Published: October 2019

Introduction:
Chronotropic incompetence is a common feature in patients afflicted by heart failure with preserved ejection fraction (HFpEF). However, the specific mechanisms remain unknown.

Aim:
To probe the mechanisms of chronotropic incompetence in HFpEF.

Methods:
Dahl salt-sensitive rats were fed high-salt diet (8% NaCl). Rats fed normal-salt diet (0.3% NaCl) served as controls. Echocardiography was used to confirm the development of diastolic dysfunction and preserved EF. Telemetry devices were implanted and treadmill exercise tests were performed to assess heart rate response to exercise. Sinus node recovery time (SNRT) was measured
in vivo,
and
ex vivo
optical mapping was performed in isolated sinoatrial node (SAN) tissue to measure heart rate response to isoproterenol.

Results:
HFpEF rats showed decreased E/A ratio (17%, p<0.05) and increased E/E’ ratio (40%, p<0.05) compared to controls, indicative of diastolic dysfunction, while EF remained unchanged in both groups. Resting mean blood pressure was elevated in HFpEF rats compared to controls (75%, p<0.05). HFpEF rats exhibited low chronotropic response to maximal exercise, in association with prolonged cSNRT (46%, p<0.05), indicative of abnormal SAN function. Despite unchanged baseline heart rate,
ex vivo
high-resolution optical mapping in isolated SAN tissue revealed delayed slow diastolic depolarization of spontaneous action potentials underlying the lower β-AR responsiveness, as well as slowed conduction (in association with histologically-evident increases in SA fibrosis). Next-generation RNA sequencing revealed that SAN from HFpEF rats shows numerous changes in transcripts associated with ion channels, protein kinases, and tissue remodeling. At the single SAN cell level, HFpEF rats showed decreased abundance of L-type Ca
2+
channels (Ca
v
1.3, 55%) and hyperpolarization-activated "pacemaker" channels (HCN4, 67%) by western blot; such protein changes were associated with remarkable reductions of both currents.

Conclusions:
Chronotropic incompetence in HFpEF is due, in large measure, to intrinsic abnormalities of the SAN. Among these, a depressed membrane “clock” features prominently in the underlying mechanism.
Abstract 813: Downregulation of the Cardiac Sodium Channel Nav1.5 Mediates Obesity-Induced Atrial Fibrillation
Authors: Sridhar Arvind , McCauley Mark , Hong Liang , Menon Ambili , Perike Srikanth , Zhang Meihong , Ai Xun , Yan JiaJie , Youn Seock-Won , da Silva Ivson Bezerra , Bonini Marcelo , Rehman Jalees , Darbar Dawood
Published: October 2019

Background:
Obesity increases atrial fibrillation (AF) risk, but the underlying mechanisms are unclear. Emerging evidence supports reduced cardiac Na
+
channel (Nav1.5) expression and current (

I
Na

) as one potential mechanism.

Objective:
We used an acquired (diet-induced obese, DIO) obese mouse model to test the hypothesis that obesity increases AF risk by downregulating Nav1.5.

Methods:
Weight, BP, plasma glucose, and F
2
-isoprostanes were measured in DIO mice and compared to controls. Echocardiography, EP studies, immunohistochemistry, Western blotting, cellular patch clamping and optical mapping studies were performed.

Results:
DIO (37.5±3.8) mice were heavier than controls (
24.3
±4.1;P
<0.001). AF burden was 355±383 sec in DIO mice vs. 8.3± 21.4 sec in controls (
P
<0.0001). Nav1.5 expression,

I
Na

, atrial action potential duration (APD) and conduction velocity (CV) were reduced, and F
2
-IsoPs were increased. AF burden was reduced in the DIO model after chronic treatment with MitoTEMPO antioxidant, with partial restoration of the APD90,
I
Na
and AP upstroke and reduction in F
2
-IsoPs.

Conclusions:
Mice with either acquired or genetic susceptibility to obesity are more prone to develop AF. And this is partially mediated by increased oxidative stress leading to downregulation of Nav1.5, shortened atrial APD and reduced atrial conduction velocity suggesting a possible reentrant mechanism of AF.
Abstract 816: Surgical Bilateral Stellate Ganglionectomy Reduces Mitochondrial Reactive Oxygen Species (mROS) and Prevents Sudden Cardiac Death (SCD) in Non-ischemic Heart Failure (HF)
Authors: Dey Swati , O'Rourke Brian , DeMazumder Deeptankar
Published: October 2019

Introduction:
Current therapies for SCD are limited. Bilateral sympathetic stellate ganglionectomy (
BSG
) is a promising new adjunctive therapy in patients with ischemic HF who have incessant ventricular tachyarrhythmias (
VT/VF
) despite optimal medical management. The underlying mechanisms are unknown. The effect of BSG in non-ischemic HF is unclear.

Hypothesis:
BSG prevents VT/VF and SCD in non-ischemic HF by restoring sympathovagal balance and reducing mROS.

Methods:
We used a unique pressure-overload guinea pig model that closely mimics human non-ischemic HF, including a high incidence of spontaneous VT/VF/SCD. We randomized the animals to non-failing controls, HF+Sham, or HF+BSG surgery. We used intention to treat analysis to evaluate VT/VF-free survival. We analyzed continuous ECG (VT/VF burden, heart rate variability, QT variability) and echo (cardiac function). In isolated left ventricular (LV) myocytes, we measured Ca
2+
transients and mitochondrial reactive oxygen species (mROS) using targeted ratiometric probes.

Results:
Half of the HF+Sham animals experienced SCD in 4 weeks. In contrast, BSG abolished SCD, reduced VT/VF, and increased parasympathetic tone (beyond corresponding reductions in sympathetic tone). Fractional shortening of the LV was markedly reduced in HF+Sham (0.28±0.02) vs. control (0.50±0.01), but preserved by BSG (0.45±0.02). Whereas HF+Sham increased mROS levels in LV myocytes (fractional oxidation, F
ox
: 0.53±0.015) compared to control (0.29±0.020), BSG reduced mROS (0.38±0.035). Mitochondrial antioxidant capacity, as indexed by H
2
O
2
challenge of LV myocytes, was compromised in HF+Sham (F
ox
: 0.81±0.040) compared to control (0.44±0.061), but preserved by BSG (0.48±0.023).

Conclusions:
Surgical BSG prevents SCD in non-ischemic HF by reducing mROS, decreasing sympathetic tone, and by increasing parasympathetic tone (possibly by facilitating cholinergic transdifferentiation). This promising new minimally-invasive surgical therapy may be particularly useful in patients with non-ischemic HF on optimal medical therapy who require recurrent ICD shocks to prevent SCD. Patients in the early stages of HF who are not eligible for ICDs but remain at higher SCD risk may also benefit from BSG surgery.
Abstract 820: Enoxaparin Reverses Ventricular Tachycardia and Torsades de Pointes in Rat Isolated Heart Model
Authors: Vasques Enio R , Vasques Enio R , Tersariol Ivarne L , Nader Helena B , Godoy Carlos M
Published: October 2019

Introduction:
Enoxaparin (Enox) is used in cardiovascular emergency and coronary angiography due to its effects on coagulation mechanisms. However, its effect on changing the structure of the exchange-inhibiting peptide present in the sodium-calcium exchanger, leading to the acceleration of its function and withdrawing calcium from the cell in overload situations could lead to a new therapeutic function, where the intracellular calcium overload would be deleterious, as in cardiac arrhythmias and myocardial infarction.

Objective:
To analyze the action of Enox on cardiac arrhythmias induced by ischemia and reperfusion (IR) in isolated rat hearts. Materials and method. Adult Wistar rats were sacrificed, hearts isolated and placed under perfusion in Langhendorf Model with Krebs solution (KS) and monitoring of the electrocardiogram. After 30 minutes in sinus rhythm, ischemia was performed with discontinuation of the KS for 5 min and then reconnected. They were divided into 2 groups: Control (n = 10), with IR without medication and Enox Group, with infusion of 2mg / kg, in the cannula that perfuses the heart, soon after initiation and support for 30 seconds of Ventricular Tachycardia (VT) or Torsade de Pointes (TP). In case of reversal of the arrhythmia, a new arrhythmia induction will be attempted with the same protocol. Monitoring is continued until asystole or completion scheduled for 60 minutes of experimentation.

Results:
The control group presented VT in 70% of hearts and total atrioventricular block (TAVB) in 30% of hearts, followed by asystole after 7 minutes in 100% of hearts, and no further attempt of ischemia was possible. In the Enox group, 60% of VT, 30% of TP and 10% of TAVB were observed. All arrhythmias were reversed between 10 and 15 seconds after infusion of Enox, with return and maintenance of sinus rhythm after medication. In this group, a new attempt was made to induce arrhythmia due to IR without success after 10 minutes of interruption of the arrhythmia by Enox and sinus rhythm was maintained until the interruption of the experimental protocol.

Conclusion:
Enoxaparin reverses arrhythmias triggered by IR, with its effect prolonging and protecting new events after infusion, as well as decreasing organ mortality in isolated hearts of rats.
Abstract 825: Attenuation of Cardiac Fibrosis by Scleraxis Gene Deletion Improves Pressure Overload-Induced Cardiac Remodeling
Authors: Nagalingam Raghu S , Cheung David Y , Aroutiounova Nina , Jassal Davinder S , Czubryt Michael P
Published: October 2019

Cardiac fibrosis is a significant independent risk factor for heart failure with increasing incidence. The fibrotic myocardium shows increased arrhythmogenesis, and poor pumping and relaxation due to greater tissue stiffness. A critical step in this process is the conversion of fibroblasts to myofibroblasts, which are responsible for excessive extracellular matrix (ECM) production; limiting this conversion may reduce fibrosis and restore cardiac function. We previously reported that the transcription factor scleraxis, following mechanical stretch or TGFβ signaling, is both sufficient and necessary to convert fibroblasts to myofibroblasts by direct transcriptional control of myofibroblast genes including collagens, α-smooth muscle actin and fibronectin. In a pressure overload transverse aorta constriction (TAC) mouse model analyzed by echocardiography, we found that fibroblast-specific scleraxis gene deletion prior to TAC using a tamoxifen-inducible TCF21-Cre/loxP approach attenuated both systolic (LV ejection fraction, fractional shortening) and diastolic (early and late filling velocity) dysfunction, as well as chamber dilation, despite persistent hypertrophy. Functional improvement was matched by an almost complete attenuation of cardiac fibrosis (Masson’s trichrome; qPCR and western blots for fibrillar collagens and ED-A fibronectin). Scleraxis deletion also prevented induction of the myofibroblast marker periostin, suggesting a failure of scleraxis-null fibroblasts to convert to myofibroblasts. We next tested if scleraxis deletion 4 weeks post-TAC could reverse subsequent remodeling at 8 weeks post-TAC. Adverse remodeling occurred in all animals 4 weeks post-TAC (prior to scleraxis deletion), but cardiac function and chamber dimensions subsequently declined further in scleraxis-intact animals, while scleraxis-deleted animals showed preserved or improved cardiac function and morphology. Our results demonstrate that scleraxis is required for the initiation and progression of cardiac fibrosis, and that reducing fibrosis alone improves cardiac performance and morphology even in the presence of persistent pressure overload. Scleraxis is thus an important target for anti-fibrotic therapy development.
Abstract 826: Histone Deacetylase Inhibition Improves Heart Failure With Preserved Ejection Fraction Cardiopulmonary Phenotype and Induces Metabolomic Switch
Authors: Eaton Deborah M , Wallner Markus , Berretta Remus M , Bordag Natalie , Wu Jichuan , Jeong Mark Y , Lin Ying H , Borghetti Giulia , Baker Sandy T , Zhao Huaqing , Rainer Peter P , Oyama Mark A , von Lewinski Dirk , Mohsin Sadia , Post Heiner , Magnes Christoph , Zügner Elmar , McKinsey Timothy A , Wolfson Marla R , Houser Steven R
Published: October 2019

Rationale:
Approximately 50% of heart failure patients are diagnosed with Heart Failure with preserved Ejection Fraction (HFpEF), but there are currently no effective treatments.

Hypothesis:
Treatment of a feline HFpEF animal model with a pan HDAC inhibitor, SAHA, will improve the cardiopulmonary phenotype and mediate changes in the metabolome and skeletal muscle composition.

Methods and results:
Male domestic short hair cats (n=21, age 2mo) underwent either a sham procedure (n=5) or aortic constriction (n=16) using a customized pre-shaped band causing slow progressive pressure overload during maturation. At 2-months post-banding, banded animals received either daily treatment with 10mg/kg SAHA (b+SAHA) (n=8) or vehicle (b+veh) (n=8) for 2 months. At 4 months post-banding, b+ SAHA animals had significantly reduced LV wall thickness, LA size (LA/Ao), and improved LA function (LA EF) vs. b+ veh animals (fig). Invasive hemodynamics and lung mechanics were performed after 2 months of treatment. Banded animals had significantly increased filling pressures (LVEDP), increased pulmonary arterial pressures (mPAP), decreased lung compliance and arterial oxygenation (A-aDO
2
). SAHA significantly reduced LVEDP, mPAP, and A-aDO
2
and increased lung compliance (fig). b+SAHA animals had an increase in the percentage of type 1 muscle fibers (increased oxidative capacity) compared to type 2 (fig). Blood-based metabolomics revealed SAHA-induced a metabolic shift towards increased lipolysis and mitochondrial oxidation.

Conclusion:
Treatment with SAHA improved cardiopulmonary structure and function in banded animals and caused changes in the metabolome and skeletal muscle.
Abstract 829: Endothelial Estrogen Non-nuclear Signaling Plays a Key Role in Anti-remodeling Effects via cGMP Signaling in Failing Heart
Authors: Fukuma Nobuaki , Takimoto Eiki , Ueda Kazutaka , Liu Pangyen , Li Yuxin , Noma Kensuke , Hiroi Yukio , Liao James K , Komuro Issei
Published: October 2019

Background:
Estrogen plays a significant role in cardiovascular benefits. As one of its beneficial pathways, ERα non-nuclear signaling is known to be protective for the vascular system, but its role for cardiac remodeling is unknown thus far. We previously reported cGMP pathways are one of the critical therapeutic targets for heart failure and ERα non-nuclear signaling played a pivotal role in cardioprotection and was indispensable to the therapeutic efficacy of cGMP-PDE5 inhibition. However, it is still remaining undetermined which cells are playing the key role in cardioprotective effects via ERα non-nuclear signaling.

Methods:
We generated genetically modified mouse lacking ERα in cardiomyocytes (ERα
f/f
/αMHC
Cre/+
) or endothelial cells (ERα
f/f
/Tie2
Cre/+
) and assessed the effects of chronic pressure-overload (TAC) and the efficacy of PDE5 inhibitor (PDE5i) sildenafil after TAC. In addition, for the first time we generated endothelial cell specific genet-modified mouse lacking interactions between p85α and ERα which are critical for non-nuclear signaling (ERα
KI/KI
/Tie2
Cre/+
). Cardiac functions were assessed for evaluating the effects of endothelial ERα non-nuclear singlaing to failing hearts.

Results:
Under physiological E2 status, PDE5i’s anti-remodeling effects after TAC were abrogated in ERα
f/f
/Tie2
Cre/+
(FS(%) ± SEM with vs without PDE5i 28.16 ± 3.43 vs 31.87 ± 2.30) but not in ERα
f/f
/αMHC
Cre/+
(FS(%) ± SEM with PDE5i vs without PDE5i 54.57 ± 3.418 vs 43.3 ± 1.21) consistent with hemodynamic analysis. Assessment with ERα
KI/KI
/Tie2
Cre/+
showed decreased cardiac functions even with physiological E2. Hemodynamic analysis also supported the cardioprotective effects of ERα non-nuclear signaling in endothelial cells.

Conclusion:
ERα non-nuclear reaction in endothelial cells plays an important role in cardio-protective mechanism, and endothelial estrogen signals are indispensable to the therapeutic efficacy of cGMP-PDE5 inhibition. Our results clarified one of the cardioprotective signals of estrogen and highlighted the new strategy for heart failure treatment.