bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 315: Efficacy and Safety of Rivaroxaban in
in vitro
Experiments With the Endothelial Cells
Authors: Seki Kaname , Mizuno Yosuke , Sakashita Toku , Tanno Jun , Nakano Shintaro , Muramatsu Toshihiro , Nishimura Shigeyuki , Senbonmatsu Takaaki
Published: June 2021

Aim:
Activated factor X (FXa) plays important roles in the thrombin generation and in inflammation, which is evoked during the endothelial damage. Although rivaroxaban is a selective FXa antagonist, it is one of the key therapies in ischemic heart disease, and yet its function in the state of inactivated coagulation cascade is uncertain. Rivaroxaban blocks FXa in the blood but not the tissue, while factor X is converted to FXa only when glutamic acid is changed to γ-carboxyglutamic acid by vitamin K following the intrinsic clotting factors and/or cellular injury activation. To uncover this aspect, we performed the following experiments.

Methods and results:
Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza Co., Ltd. The cells were grown to 80% confluence and were treated with rivaroxaban (100nM, 500nM, 1000nM, 2000nM respectively) without FXa stimulation for 4 h, 10 h or 24 h. Cells and medium were collected and then their RNA was extracted from the cells. The qPCR of MCP-1, PAR1-4 and the DNA micro arrays (The GeneChip Human Gene 2.0 ST Array, Affymetrix) were performed. There was neither increased nor decreased gene expression significantly in either experimental time course of the qPCRs or the the DNA micro arrays. The ELISA assay of MCP-1 with medium showed non-activated MCP-1. As a next step, cells were treated with 100nM FXa and with/without rivaroxaban in same time course, and cells and medium were collected for further experiments. FXa evoked induction of mRNA levels for several pro-inflammatory cytokines including MCP-1 maximally at 4h, whereas MCP-1 was maximally evoked at 24 h in ELISA assay. Interestingly rivaroxaban inhibited both in all time course, at 4 hour inflammatory phase and at 24 hour inflammatory phase.

Conclusion:
Collectively, these results suggest that rivaroxaban may be safe in the inactivated coagulation state, and has the efficacy to attenuate the endothelial damage evoked by FXa and by pro-inflammatory cytokine genes.
Abstract 316: Engineering Nanomaterial Morphology for Targeting Immune Cells Within Atherosclerotic Lesions
Authors: Yi Sijia , Liu Yugang , Allen Sean , Du Fanfan , Li Xiaomo , Ouyang Brian , Scott Evan
Published: June 2021

Atherosclerosis is a chronic vascular inflammatory disease, in which several types of immune cells have been identified as playing important roles. Nanomaterials can function as powerful theranostic platforms for diagnostic imaging and controlled delivery of therapeutics in atherosclerosis. Here, we present a detailed investigation into the effects of morphology on the
in vivo
biodistribution of nanomaterials in naïve mice following intravenous injection. We applied these findings towards the targeting of diverse immune cells within the lesions of atherosclerotic mice. Three different nanostructures of the same surface chemistry were assembled from poly(ethylene glycol)-
bl
-poly(propylene sulfide) (PEG-
bl
-PPS) block copolymers: micelles (30 nm), vesicles (120 nm) and filomicelles (50 nm diameter by micron length). To assess the effects of the different morphologies, a multimodal approach was utilized that included 1) near infrared fluorescence (NIRF) imaging to quantify organ targeting, and 2) fluorescent polymer conjugation for subsequent flow cytometric analysis of uptake by immune cells. Of note, vesicles were exceptionally efficient at targeting the spleen and were associated with up to 85% of plasmacytoid dendritic cells. Micelles were associated with up to 90% of macrophages in the liver, and filomicelles were most effective at avoiding uptake by the cells of the mononuclear phagocyte system. Due to their enhanced uptake by dendritic cell subsets relative to other nanostructures, vesicles were selected for targeting cells within aortic lesions of atherosclerotic LDL
-/-
mice. In addition to associating with macrophages and eosinophils, vesicles were found to target significantly higher percentages of atheroma-resident dendritic cells (25%). In conclusion, differences in morphology can drastically change the biodistribution of nanomaterials at both the organ and cellular level. The ability to target or avoid phagocytic cell subsets will enhance current and future theranostic strategies. Furthermore, the targeting of dendritic cells by vesicular nanostructures within atherosclerotic lesions opens new avenues for immunotherapies in cardiovascular disease.
Abstract 319: Structural Reorganization of Cardiac Transcription Factories Mediates Transcriptional Changes in Response to Stress
Authors: Karbassi Elaheh , Rosa Garrido Manuel , Chapski Douglas J , Wu Yong , Monte Emma , Vondriska Thomas M
Published: June 2021

The heart’s response to stress entails precise gene expression changes to affect the metabolic and structural features of the cardiomyocyte. The changes in gene expression are mediated by structural alterations in the packaging of the genome. However, the manner in which the three-dimensional architecture of the genome is established is unknown. In non-cardiac cells, genes that are actively transcribed are thought to reside in transcriptionally permissive compartments called transcription factories. The structural principles for achieving cardiac-specific transcription are not understood. We sought to understand the functional nature of cardiac transcription factories: whether they are stable structures (to which genes move in and out of) or are transiently formed around genes in response to cardiac stimuli. Using 5-fluorouridine incorporation into nascent RNA, we quantified changes in RNA polymerase II-mediated transcription in cardiomyocytes upon hypertrophic stress. Furthermore, we characterized the spatial distribution of transcription factories, marked by RNA polymerase II, from adult mice subjected to pressure overload. Using super-resolution microscopy, our analyses revealed reorganization of RNA polymerase II, evidenced by a significant increase in the distance between clusters (130nm in sham to 132.5nm in failing hearts, p=0.02) and a 38% increase in cluster intensity in failing hearts. To understand regulation of cardiac gene expression, we used DNA fluorescence
in situ
hybridization to map the nuclear position of the gene for SERCA2a (atp2a2), which is down regulated in disease. In failing hearts, we measured increased association of atp2a2 with the nuclear envelope (0/159 loci in sham to 11/278 loci in failure) and increased colocalization with heterochromatin (53/160 loci in sham versus 139/290 loci in failure), providing a structural mechanism for the decrease in SERCA2a expression. In contrast, atp2a2 positioning in the liver remained unaffected, with the majority of loci colocalizing with heterochromatin. These findings show that RNA polymerase II is redistributed to affect transcriptional programming and characterize for the first time the structural rearrangements in chromatin that underpin cardiac pathology.
Abstract 322: Postnatal Islet-1 Cardioblasts Are of Neural Crest and Not Second Heart-Field Lineage
Authors: Hatzistergos Konstantinos E , Hare Joshua M
Published: June 2021

Introduction:
The transcription factor Islet-1 (Isl1) is expressed in cardiac mesodermal and neural crest (CNC) lineages during cardiogenesis. A pool of Isl1
+
cells persist in the perinatal heart (Isl1
+
CPCs), some of which are touted as residual second heart-field (SHF)- derived cardioblasts. However, direct lineage-tracing evidence, supporting a SHF over a CNC origin of Isl1
+
CPCs, are lacking.

Hypothesis:
Isl1
+
CPCs are of CNC and not SHF lineage.

Methods:
The
Isl1-nLacZ,
Wnt1-Cre;tdTomato
and
Wnt1::FlpE;RC::Fela
mice, and iPSCs derived from
Wnt1-Cre;tdTomato
mice (iPSC

Wnt1

) were employed to lineage-trace Isl1
+
CPCs.

Results:
Temporal analysis of
Isl1-nLacZ
embryos illustrated a transient, stage-specific reporter gene activity in mesendodermal and neuroectodermal cells. Particularly, at embryonic day (E)9.5, reporter gene activity (x-gal), was strong in the outflow tract (OFT), and exhibited a weak, spotty pattern in the heart. At E12.5, x-gal was strong in the neural tube (NT); reduced in the OFT; and undetectable in the heart. Isl1 immunohistochemistry (IHC) illustrated similar activity of x-gal with endogenous Isl1 expression. Importantly, x-gal remained permanently undetected in the heart. Isl1 IHC in E12.5
Wnt1-Cre;tdTomato
and
Wnt1::FlpE;RC::Fela
embryos indicated that Isl1
+
cells in the OFT and NT colocalized with
Wnt1
reporter transgenes. At E18.5 and postnatal day 1 (PN1), Isl1
+
cells were exclusively of
Wnt1
lineage, indicating that Isl1
+
CPCs are CNC- and not SHF-derived. Since CNCs minimally contribute cardiomyocytes in mammals, a presumptive full cardiomyogenic capacity of Isl1
+
CPCs potentially contrasts with a CNC origin. To address this controversy, we differentiated iPSC

Wnt1

toward the CNC lineage, using a previously established embryoid body (EB) differentiation protocol involving transient BMP antagonism. At ~EB-day 9, tdTomato
+
/Isl1
+
CNCs emerged, which progressively differentiated into spontaneously beating, Nkx2.5
+
cardiomyocytes.

Conclusions:
Our findings clarify that Isl1
+
CPCs are of CNC and not SHF origin, and suggest a novel role of the mammalian CNC, as a contributor of long-lived myocardial progenitors that could be targeted for heart-related therapeutic purposes.
Abstract 351: Allogeneic C-kit
+
Cardiac Progenitor Cells are Immune Tolerant With Strong Efficacy for Cardiac Functional Recovery
Authors: Datla Srinivasa Raju , Guo Yin , Mishra Rachana , Chen Ling , Saha Progyaparamita , Sharma Sudhish , Bigham Grace , Jack Godly , Kaushal Sunjay
Published: June 2021

Background:
A recent clinical trail using adult autologous c-kit
+
cardiac progenitor cells (c-kit
+
CPCs) have demonstrated cardiac functional recovery in myocardial ischemic patients. This autologous approach of ex vivo expansion of c-kit+ CPCs is expensive and time consuming. The immune tolerant properties of c-kit
+
CPCs have not been investigated as an alternative clinical strategy. In this study, we compared allogeneic and syngeneic c-kit
+
CPCs immunogenic tolerance and cardiac recovery potentials using divergent inbred rat strains in a myocardial infarction (MI) model.

Methods and Results:
We isolated rat c-kit
+
CPCs (rc-kit
+
CPCs) from male Wistar Kyoto (WKY) rat hearts and human c-kit
+
CPCs (hc-kit
+
CPCs) from adult coronary artery bypass graft patient’s right atrial appendage. In vitro, both rat and human c-kit
+
CPCs expressed MHC class I but not class II or co-stimulatory molecules CD80 and CD86. In mixed lymphocyte reaction (MLR) assay, allogeneic and syngeneic rc-kit
+
CPCs elicited minimal T-cell proliferation. In vivo, intra-myocardial administration of WKY rc-kit
+
CPCs after creating MI condition, elicited minimal local, systemic and humoral inflammatory response in allogeneic (Brown Norway (BN) female rats) and syngeneic (WKY female rats) groups compared to the xenogeneic (hc-kit
+
CPCs in BN male rats). In addition, the allogeneic and syngeneic rc-kit
+
CPCs groups significantly recovered cardiac function (EF, SV, and CO) compared to the xenogeneic c-kit
+
CPCs group, which had no functional recoverability. Histological analysis further revealed that allogeneic and syngeneic rc-kit
+
CPCs groups had significantly decreased scar size by increasing endogenous cardiomyocyte proliferation, c-kit
+
CPC recruitment and increased neoangiogenesis.

Conclusion:
Allogeneic c-kit
+
CPCs are immune tolerant and produce minimal inflammatory response while maintaining their cardiac recovery potentials in immunologically divergent species. Our observation, for the fist time, reports the minimal immunogenic response by c-kit
+
CPCs and strongly supports the development of an allogeneic c-kit
+
CPC strategy for clinical trial applications.
Abstract 353: P2Y
2
Nucleotide Receptor Overexpression: Letting Blind Cardiac Progenitor Cells ‘See’ Again
Authors: Khalafalla Farid , Greene Steven , Nguyen Jonathan , Ilves Kelli , Norman Benjamin , Alvarez Roberto J , Sussman Mark
Published: June 2021

Heart failure (HF) is a leading cause of death in the US due to limited capability of adult mammalian heart to regenerate after myocardial infarction (MI). Autologous stem cell therapy holds promise for promoting regeneration of injured heart but stem cells derived from diseased organs exhibit poor proliferative, migratory and survival capabilities. Empowering cardiac-derived progenitor cells (CPC) with prosurvival genes has been attempted. However, molecular mechanisms by which stem cells detect stress signals to subsequently initiate appropriate regenerative responses are poorly understood. In this regard, purinergic receptors represent a major detector for extracellular nucleotides released during injury/stress and serve as an intracellular platform harboring numerous signaling pathways that regulate proinflammatory and regenerative responses required for the healing process. Despite the established roles of purinergic signaling in cardiovascular diseases, it has not been well-defined in CPCs. This study shows, for the first time, that the majority of P2 purinergic receptors are expressed and exhibit functional responses to ATP and UTP in human CPCs (hCPC) isolated from HF patients. The G protein-coupled P2Y
2
R is a pivotal stress detector that senses ATP and UTP accumulated in extracellular space after injury and mediates regenerative responses in various injury models, including MI model, and in stem cells from diverse origins. Interestingly, hCPCs with relatively slower growth kinetics and enhanced senescence show dramatic decreases in P2Y
2
receptor (P2Y
2
R) expression compared to fast-growing hCPCs, consistent with our hypothesis that overexpressing P2Y
2
R enables diseased hCPCs to better detect stress stimuli and react with the proper regenerative responses. Along this line, P2Y
2
R stimulation with UTP enhances hCPC proliferation and migration. Interestingly, preliminary results demonstrate that UTP treatment induces YAP activation and nuclear shuttling. Moreover, inhibition of YAP/TEAD interaction impairs UTP-induced proliferation and migration revealing a novel link between extracellular nucleotides released during cardiac ischemia and Hippo signaling that has been recently implicated in cardiac regeneration.
Abstract 354: Optimization of Extracellular Vesicles Isolation From Cardiac Mesenchymal Cells
Authors: Farid Talha A , Pathan Asif R , Khan Abdur R , Nasr Marjan , Li Hong , Muthusamy Senthilkumar , Wysoczynski Marcin , Bolli Roberto
Published: June 2021

Background:
Cell therapy and cell derived extracellular vesicles (EVs) are promising treatment options for ischemic cardiomyopathy. Despite this increasing interest, the isolation of EVs is not standardized. The purpose of this study was to compare two most common methods of EV isolation, ultracentrifugation (UC) and polyethylene glycol precipitation (PEG); and also to test the hypothesis that PEG precipitation followed by UC (PEG+UC) offers a more efficient method of EV isolation compared to PEG precipitation or UC alone.

Methods:
EVs were isolated from mouse cardiac mesenchymal cells by either PEG, UC (2 cycles at 100,000g, 1 hour each), or PEG+UC (100,000g for 1 hour). Size analysis, protein content measurements and western blots (WB) were performed.

Results:
PEG precipitation yielded three distinct population of particles (~20-60nm, ~90-500nm & >1000nm – Figure 1A). UC and PEG+UC provided one distinct population of EVs (90-500nm). With PEG precipitation, the highest concentration of particles obtained were in the range of 20-60nm (~99%, Figure 1B). With PEG+UC, these smaller particles were cleared yielding an uncontaminated EV population (90-500nm). PEG precipitation yielded highest content of protein followed by PEG+UC (Figure 1C). WB demonstrated weak expression of EV markers with PEG precipitation and enriched markers with UC & PEG+UC (Figure 1D).

Conclusion:
PEG precipitation yields least pure population of EVs with highest protein contamination, likely from precipitation of soluble proteins. UC provides pure EV population with low yield. PEG precipitation followed by UC provides the highest yield of EVs while maintaining EV purity.
Abstract 360: Implantation of an Induced Pluripotent Stem Cell Derived Cardiomyocyte Tissue Engineered Patch Improves Left Ventricular Function and Electro-mechanical Coupling in Rats With Heart Failure
Authors: Lancaster Jordan J , Repetti Giuliana , Pandey Amitabh , Weigand Kyle , Bahl Joseph J , Juneman Elizabeth , Goldman Steven
Published: June 2021

Background:
Chronic Heart Failure (CHF) is the leading cause of hospital readmissions and mortality in the US. Here we report the effects of surgically delivering a human bioengineered patch of human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CMs) and fibroblasts on left ventricular (LV) function in rats with CHF. We evaluate improvements in LV systolic and diastolic function, electromechanical coupling and gene expression after patch implantation.

Methods:
Adult male Sprague-Dawley rats underwent left coronary artery ligation and were randomized to Sham (N=8), CHF (N=8-21), and CHF+hiPSC-CM patch (N=20-24). Heterogeneous hiPSC-CMs were seeded and co-cultured onto a vicryl matrix embedded with human dermal fibroblasts. Echocardiography was performed at 3 and 6 weeks post-randomization. Hemodynamic pressure measurements were performed at 6 weeks post-ligation with Millar solid state micromanometer pressure catheters. Open chest Electrophysiologic (EP) mapping was performed at 6 weeks post ligation. Gene expression was evaluated through qRT-PCR.

Results:
48 hours into culture hiPSC-CMs patches displayed synchronized and spontaneous contractions which developed in robustness over time. At maximal robustness, contractions were visualized across the full thickness of the construct. Contractions were recorded at 36
+
5 beats BPM. Three weeks after patch implantation (6 weeks post ligation) the hiPSC-CM patch decreased (P<0.05) LV EDP (45%), Tau (29%), E/e’ (23%) and increased (P<0.05), e’/a’ (36%) with trending improvements in EF (14%) and e’ (20%). EP studies show electro-mechanical coupling between the patch and the native myocardium with normal activation through the patch and increases (P<0.05) voltage amplitude in CHF versus hiPSC-CM patch treated rats (1±0.5 mV vs 6±1.5mV). Rats treated with the hiPSC-CM patch showed significant (P<0.05) fold expression of Cx43 (3.3), ANG-1 (13.63), VEGF (3.8), βMYH7 (6.4) and IGF-1 (22.9) versus control.

Conclusion:
Cardiac patch implantation with hiPSC derived cardiomyocytes is an effective and feasible method of treating CHF with improvements in systolic function, diastolic function, and electro-mechanical coupling in rats with CHF.
Abstract 380: Impairment of Human Microvascular Function Induced by β-amyloid and Cardiovascular Risk Factors
Authors: Truran Seth , Karamanova Nina , Anderson Jenna , Serrano Geidy , Beach Thomas , Franco Daniel , Burciu Camelia , Reaven Peter , Migrino Raymond
Published: June 2021

Alzheimer’s disease (AD) and aging-related dementia are associated with early vascular dysfunction (VD) and increased cardiovascular metabolic risk factors (CVRFs). We showed that β-amyloid peptide (Aβ) induce human leptomeningeal arteriole (LMA) endothelial dysfunction and oxidative stress and peripheral adipose arterioles (AA) may be useful surrogates to assess central cerebrovascular response. It is not known whether combined Aβ and CVRFs cause synergistic VD. We tested the hypothesis that 1) Aβ and CVRFs (saturated fatty acid [palmitic acid, PA]) and hyperglycemia [HG]) induce VD and combined, would cause synergistic injury and 2) AA would parallel LMA responses.

Methods:
LMA were collected following rapid autopsy from brain donors. Abdominal AA were collected from volunteers without AD or vascular disease undergoing herniorrhaphies. Arterioles were pressurized. Dilator responses to acetylcholine (10
-9
-10
-4
M) and papaverine (10
-4
M) were measured at baseline and following 1-hour exposure to Aβ42 (0.5 or 2 μM), PA (150 μM), HG (33 mM), Aβ+PA or Aβ +HG.

Results:
See figure. When only LMA with normal baseline response were included, Aβ (0.5 and 2 μM) and PA, but not HG, showed impaired dilation to acetylcholine versus control.

Conclusions:
Aβ induced endothelial dysfunction in human LMA and AA and smooth muscle dysfunction in AA. Co-treatment with PA and HG did not result in additive/synergistic injury at the doses used. Similar and differential responses to Aβ±CVRFs between AA and LMA point to need for further studies to assess if peripherally obtained AA recapitulate vascular physiology of cerebrovascular arterioles sufficiently to serve as experimental surrogate.
Abstract 383: Immunotherapy With
Trypanosoma cruzi
Genes Prevents the Severity of Heart Damage in Experimental Chagasic Dogs Evaluated by Electrocardiography and Macroscopic Findings at Necropsy
Authors: Rodríguez-Morales Olivia , Carrillo-Sánchez Silvia C , Rios-Castro Martha , Arce-Fonseca Minerva
Published: June 2021

Chagas disease is a chronic and potentially lethal disorder caused by the parasite
Trypanosoma cruzi.
The main importance of this parasitosis is its high prevalence, its wide geographical distribution and the great economic losses because it affects young adults in productive life causing deterioration of cardiovascular health and sudden death in apparently healthy people. Currently, there is no effective treatment, since only two drugs with trypanocidal activity can cause cure in 80% of patients only if they are treated promptly during the acute phase of the disease. The aim of this study was to test whether immunotherapy with parasite DNA is able to avoid, reduce or delay the chagasic cardiomyopathy in experimentally infected dogs. Thirty Beagle adult dogs were used; five groups were infected with 3,500 metacyclic trypomastigotes of
T. cruzi
H8 strain/kg, two weeks later 500 ug of plasmid DNA containing
TcSP
,
TcSSP4
and the mixture of both genes were injected thrice at 2-week intervals. Empty vector and saline solution were used as controls. Prior to infection/immunotherapy electrocardiographic features were recorded in all dogs as well as at 3, 6 and 12 months later. Hearts, spleens and lymph nodes were collected after euthanasia to determine cardiomegaly, splenomegaly and lymphadenomegaly, respectively, in infected/treated dogs. The results were that the DNA vaccines used as immunotherapy had moderate efficacy in preventing cardiac complications or delay its onset associated with chronic chagasic cardiomyopathy evaluated by electrocardiography such as left ventricle enlargement, left and right bundle branch block and ischaemia.
T. cruzi
genes administered separately prevented cardiomegaly. In conclusion, the use of DNA with
TcSSP4
and
TcSP
genes is an alternative use of new therapeutic tool against CD, which in recent years has taken off by gathering efforts of researchers and physicians from several countries, not only in Latin America.
Abstract 395: Voluntary Exercise Enhances Cardiac Growth in Response to Chronic β-Adrenergic Receptor Stimulation
Authors: Luckey Stephen W , McLaughlin Nathan , Soo Sherilynn
Published: June 2021

Exercise training protects the heart against the adverse effects of cardiovascular disease. Recent studies have identified a number of cardiac adaptations including the activation of hypertrophic signaling pathways unique to exercise. However, the underlying mechanisms by which exercise confers cardioprotection are not entirely understood. This study aims to fill this gap by examining the role of voluntary exercise in the context of chronic β-adrenergic receptor stimulation. To do this, we developed a novel experimental model in which nine-week-old female and male CB6F1 hybrid mice were subjected to 5 weeks of voluntary wheel running (EX) or housed under sedentary conditions (SED). For the final two weeks, mice were administered either vehicle (VEH) or isoproterenol (ISO, 30mg/kg/day) via an osmotic pump. As expected, we found that ISO significantly increased heart size in sedentary females and males (SED+ISO) compared to sedentary mice receiving VEH (SED+VEH). Consistent with previously published data, exercise capacity was also greater in females compared to males with regards to running duration and distance regardless of the experimental group. While exercise capacity was not affected by the administration of VEH, mice receiving ISO (EX+ISO) exercised significantly less. Cardiac growth in EX+VEH mice was significantly increased in both females and males compared to their respective SED+VEH counterparts. Importantly, EX+ISO females and males have significantly larger hearts than their respective SED+ISO cohorts. Moreover, EX+ISO mice also exhibited greater increases in cardiac size as compared to their respective EX+VEH counterparts. Thus, we conclude that there appears to be an additive effect of voluntary exercise and ISO administration in both females and males in terms of cardiac growth. These preliminary data are in contrast to previously published data which found that controlled exercise programs reduced cardiac hypertrophy under conditions of chronic β-adrenergic receptor stimulation. We are currently investigating the processes that lead to the larger heart sizes in the EX+ISO mice and aim to better understand the underlying mechanisms of exercise-mediated cardioprotection.
Abstract 409: Matrix Metalloproteinase-13 Inhibition is Protective in a Pressure Overload Model of Heart Failure
Authors: Schafer Allison E , Valiente-Alandi Iñigo , Blaxall Burns C
Published: June 2021

Heart failure (HF), the leading cause of morbidity and mortality in the United States, is characterized by pathologic remodeling, fibrosis and deteriorating cardiac function. Cardiac fibrosis occurs due to imbalanced production and degradation of extracellular matrix (ECM) proteins. Cardiac fibroblasts (CF) are largely responsible for the secretion of ECM proteins in the heart, and upon injury, transition to a migratory and proliferative myofibroblast (MF) phenotype, leading to excess ECM deposition. Elevated expression of matrix metalloproteinases (MMPs), proteolytic enzymes responsible for degradation of the ECM, is common in HF. Specifically, MMP13 is known to be upregulated in human HF patients. Therefore, we hypothesized that MMP13 plays an important role in pathologic cardiac remodeling, and that inhibition of MMP13 would prevent the development of HF in a pressure overload model, transverse aortic constriction (TAC). Mice were subjected to TAC and treated with the MMP13 inhibitor, WAY170523 (WAY), or vehicle 4 weeks post-TAC until 12 weeks post-TAC. Mice treated with WAY display decreased cardiac hypertrophy and preserved cardiac function compared to vehicle treated mice. WAY treatment may also attenuate interstitial and perivascular fibrosis as well as expression of pro-fibrotic genes. To determine the effect of MMP13 inhibition in cardiac cells, CF and MF were isolated from healthy mice or mice 5 days post-ischemia/reperfusion injury, respectively, and treated with WAY. MMP13 inhibition led to decreased CF invasion but did not affect migration, proliferation or adhesion. Interestingly, inhibition of MMP13 in MF attenuated migration, proliferation and invasion. Moreover, WAY treatment reduced collagen and fibronectin deposition in the ECM of MF. MMP13 inhibition also appeared to decrease Angiotensin II-induced hypertrophy in ventricular cardiomyocytes (CM). These data suggest a role for MMP13 in pressure overload-induced HF, CM hypertrophy and CF behavior. MMP13 inhibition after injury may attenuate cardiac hypertrophy as well as the CF to MF transition, leading to decreased cardiac fibrosis and improved cardiac function. Further understanding of the role of MMP13 could lead to a novel therapeutic target in the treatment of HF.
Abstract 42: Alternative Autophagy Plays a Role in the Effect of RAS-inhibition on Arterial Senescence and Arteriosclerosis
Authors: Ikeda Yoshiyuki , Iwabayashi Masaaki , Sasaki Yuichi , Akasaki Yuichi , Higuchi Koji , Miyata Masaaki , Ohishi Mitsuru
Published: June 2021

Background:
Autophagy is a system to maintain cell homeostasis. Besides the conventional form of autophagy (CA) depending on LC3, the presence of an alternative form of autophagy (AA) depending on Ulk1 and Rab9 has recently been reported. Renin-angiotensin-aldosterone (RAS) signal accelerates arterial senescence and develops arteriosclerosis. Whereas inhibition of RAS signal retards arterial senescence and arteriosclerosis, it remains to be elucidated how autophagy is involved in the effect of RAS-inhibition on arterial senescence and arteriosclerosis.

Methods:
We used double knockout mice (DKO) of angiotensin II type Ia receptor KO and apolipoprotein E KO (apoE KO). Aortic senility and atherosclerotic lesions were measured using senescence associated beta galactosidase (SA-beta Gal) and Oil Red O staining, respectively. Molecular analysis was also performed.

Results:
The degree of aortic senescence and atherosclerotic lesions in DKO were significantly lower than those of apoE KO, although there was no significant difference in serum lipids profile between them. Aortic expressions of p21 and PAI-1, the level of reactive oxygen species, mitochondrial function and the amount of damaged mitochondria in DKO were significantly lower than in apoE KO. These results suggest that RAS-inhibition retards arterial senescence and arteriosclerosis possibly through the mitochondrial quality control. Whereas electron microscopic analysis revealed the number of autophagosome containing mitochondria are higher in DKO than in apoE KO, there was no difference in the ration of LC3II/LC3I between them, suggesting that CA is not involved in this model. On the other hand, aortic expressions of the ratio of p-Ulk1 at Ser555/total Ulk1 and Rab9 were significantly higher in DKO than apoE KO, suggesting that AA is induced in DKO.

Conclusion:
Alternative autophagy, but not conventional autophagy, plays a crucial role in the effect of RAS-inhibition on arterial senescence and arteriosclerosis.
Abstract 421: Activated Cardiac Myofibroblasts Can Revert Back to Become Resident Fibroblasts Upon Injury Cessation
Authors: Kanisicak Onur , Karch Jason , Khalil Hadi , Maliken Bryan , Molkentin Jeffery D
Published: June 2021

Resident cardiac fibroblasts (CFs) are potential therapeutic targets in treating heart failure given the prominent role that fibrosis plays in this disorder. CFs directly convert to myofibroblasts (MFs) with injury where they mediate both adaptive wound healing after acute myocardial infarction as well as long-standing fibrosis during chronic disease states. However, the fate of activated MFs after injury recovery or when an infarction scar is stabilized remains poorly understood, in part because the field has lacked a definitive strategy for identifying and tracing MFs and CFs in vivo. To address this issue we recently generated a novel mouse model that permits lineage tracing of all MFs in the heart after injury or stress stimulation, which we used to address the fate of MFs after injury resolution. MFs were lineage traced with a tamoxifen inducible periostin allele knockin of the MerCreMer cDNA (PostnMCM), with a Rosa26-eGFP dependent reporter. PostnMCM x R26-eGFP mice were transiently injured with the combined infusion of angiotensin II and phenylephrine (Ang/PE) for 2 weeks, during which time tamoxifen was also given to trace all newly formed MFs. Mice were then allowed to “rest” for 2 weeks or longer with no Ang/PE as the fibrotic response regressed, and the fate of the eGFP
+
cells was assessed. The data show that immediately after 2 weeks of Ang/PE infusion nearly all the eGFP+ periostin lineage-traced myofibroblasts were αSMA positive and have an activated myofibroblast gene expression profile. However, when the fibrotic response regressed weeks later, a number of periostin-lineage traced eGFP+ cells were still present in the heart and these cells showed a phenotypic and molecular reversion back to CFs with a loss of myofibroblast marker genes. These results suggest that CFs are very unique cell types that can differentiate to MFs then back again to resident CFs.
Abstract 430: Spectral Changes Caused by Radiofrequency Ablation of Cardiac Tissue
Authors: Aljishi Mohammed , Asfour Huda , Chahbazian Tigran , Muselimyan Narine , Swift Luther , Mercader Marco , Sarvazyan Narine
Published: June 2021

Persistent atrial fibrillation is commonly treated using an endoscopic catheter to eliminate anomalous sources of cardiac activity via thermal ablation. However, the procedure lacks real-time feedback. Newly developed radiofrequency ablation (RFA) catheters include a fiberoptic bundle through which visual information of tissue conditions may be collected offering an opportunity to reveal subtle differences in tissue physiology. Currently little is known about the spectral changes caused by RFA. We hypothesized that by comparing spectral changes in various areas in the heart before and after RFA, optical signatures can be used to distinguish healthy cardiac tissue from thermally ablated tissue. Excitation emission matrices (EEM) were acquired from excised porcine hearts (300-600nm). Distinct EEMs were collected from the endocardium of the left atria, ventricle, and aorta. Additionally, the fluorescence and reflectance profiles of each tissue were altered by thermal ablation. In the ventricular muscle, a reduction in the NADH fluorescence peak (360/460nm excitation/emission maxima) was most prominent. While in the aorta, collagen and elastin fluorescence peaks fused and broadened upon ablation. Changes in atrial tissue included a drop in NADH fluorescence and an overall increase in reflectance. The latter is likely caused by thermal coagulation of heme-containing proteins such as myoglobin and a weaker absorption within the Soret band. We concluded that optical signals revealed by EEMs offer quantitative information that can be used to develop diagnostic catheters, including hyperspectral imaging protocols to discern spectral changes elicited by RFA treatment.
Abstract 44: TAK1 Regulates Caspase 8 Activation and Necroptotic Signaling via Multiple Cell Death Checkpoints
Authors: Guo Xaioyun , Yin Haifeng , Chen Yi , Li Lei , Li Jing , Liu Qinghang
Published: June 2021

Necroptosis has emerged as a new form of programmed cell death implicated in a number of pathological conditions such as ischemic injury, neurodegenerative disease, and viral infection. Recent studies indicate that TGFβ-activated kinase 1 (TAK1) is nodal regulator of necroptotic cell death, but the underlying molecular regulatory mechanisms remain elusive. Here we reported that TAK1 regulates necroptotic signaling as well as caspase 8 activation through both NFκB-dependent and -independent mechanisms. Inhibition of TAK1 promoted TNFα-induced necroptosis through the induction of RIP1 phosphorylation/activation and necrosome formation, in the presence of ongoing caspase activation. Further, inhibition of TAK1 triggered two caspase 8 activation pathways through the induction of RIP1-FADD-caspase 8 complex as well as FLIP cleavage/degradation. Mechanistically, our data uncovered an essential role of the adaptor protein TRADD in caspase 8 activation and necrosome formation triggered by TAK1 inhibition. Moreover, ablation of the deubiqutinase CYLD prevented both apoptotic and necroptotic signaling induced by TAK1 inhibition, whereas deletion of the E3 ubiquitin ligase TRAF2 had the opposite effect. Finally, blocking the ubiquitin-proteasome pathway prevented the degradation of key necroptotic signaling proteins and necrosome formation. Thus we identified novel regulatory mechanisms underling the critical role of TAK1 in necroptotic signaling through regulation of multiple cell death checkpoints. Targeting key components of the necroptotic pathway (e.g., TRADD and CYLD) and the ubiquitin-proteasome pathway may represent novel therapeutic strategies for pathological conditions driven by necroptosis.
Abstract 445: Characterizing The Cardiac Acetylome During Exercise
Authors: Liu Laura , Keshishian Hasmik , Liu Xiaojun , Svinkina Tanya , Lerchenmüeller Carolin , Rowe Glenn C , Chan Mun C , Carr Steven A , Rosenzweig Anthony
Published: June 2021

Recent advances in mass spectrometry have led to in-depth characterization of both tissue and cellular acetylation profiles (or “acetylomes”). These studies reveal the abundance and importance of this post-translational modification not only in transcriptional control, but also in many signaling and metabolic pathways. In the heart, acetylation has been found to play a role in cardiac proliferation, differentiation, and hypertrophy in both developmental and pathological settings. To examine the role of acetylation in exercise-induced cardiac hypertrophy, we studied the expression of enzymes regulating these processes and then undertook a comprehensive proteomic analysis of cardiac proteome and acetylome in hearts from sedentary and exercised mice. We first characterized the expression profiles of 8 acetylases (MYST1-4, GCN5, PCAF, EP300, CREBBP) and 18 deacetylases (HDAC1-11 and SIRT1-7) in four different exercise protocols, including swimming, voluntary wheel run, acute treadmill run, and long-term treadmill run in aged mice. Then, using a proteomics workflow with greater sensitivity and higher coverage recently developed at the Broad Institute, we determined the acetylome of the swim exercised mouse heart (n=5) in comparison to sedentary animals (n=4). We concurrently assessed proteome and phospho-proteome profiles in the same set of samples. Confirmation of the presence of physiological hypertrophy and increased cardiac function with exercise was determined with echocardiography and morphometric analysis. With this method, we identified 4159 acetylation sites and 19,402 phosphorylation sites on 6615 proteins. Acetylated proteins were found to be involved in a diverse array of processes, but most prominently in metabolism and muscle contraction. Phospho-proteome analysis has highlighted the interplay and crosstalk between acetylation and phosphorylation. In summary, the cardiac acetylome is dynamically regulated by exercise and demonstrates major alterations in metabolic pathways. Understanding how physiological hypertrophy affects acetylation and the activity of downstream factors may provide novel insights into the cardioprotective role of exercise.
Abstract 455: FGF23-induced Cardiac Hypertrophy is Reversible
Authors: Grabner Alexander , Singh Saurav , Schramm Karla , Yanucil Christopher , Czaya Brian , Hare Joshua , Faul Christian
Published: June 2021

Left ventricular hypertrophy (LVH) is a common feature of cardiovascular disease in chronic kidney disease (CKD) and affects up to 90% of patients by the time they reach dialysis. Serum levels of fibroblast growth factor (FGF) 23 continuously rise as patients progress to renal failure. We have previously shown that FGF23 can activate FGF receptor (FGFR) 4 and the PLCgamma/calcineurin/NFAT signaling cascade in cardiac myocytes and induce hypertrophy in vitro and in vivo. Administration of an isoform-specific FGFR4 blocking antibody in the 5/6 nephrectomy rat model of CKD immediately after surgery protects rats from developing LVH, and delivery of a pan-FGFR blocker in CKD rats two weeks after surgery reverses LVH. To further study the reversibility of cardiac FGF23 effects, we elevated serum FGF23 levels in mice by administration of a high phosphate (2%) diet for three months. Animals developed LVH, as evident by significantly increased LV wall thickness and myocyte cross sectional area. When mice were switch from high phosphate to normal chow, the LVH phenotype resolved and cardiac parameters were comparable to those of mice that constantly received a normal diet. Furthermore, isolated cardiac myocytes recovered within 24 hours from FGF23-induced hypertrophy upon removal of FGF23. Finally, the FGFR4 blocking antibody was capable of reversing FGF23-induced hypertrophy in vitro. Our data indicate that FGF23-induced LVH is reversible and treatable. Interfering with FGF23/FGFR4 signaling in the heart might provide a novel therapeutic strategy to tackle cardiac injury in CKD. We propose that progression and reversibility of cardiac injury might depend on the duration of cardiac FGF23/FGFR4 activation.
Abstract 459: Oxidized CaMKII Causes Atrial Fibrillation Susceptibility in a Diabetic Mouse Model
Authors: Mesubi Olurotimi O , Rokita Adam G , Chen Biyi , Song Long-Sheng , Wehrens Xander H , Anderson Mark E
Published: June 2021

Background:
Atrial fibrillation (AF) and diabetes mellitus (DM) are major, unsolved public health problems. DM is a known risk factor for AF, and both are associated with increased reactive oxygen species (ROS), suggesting a ROS responsive disease signal could be a mechanistic link between them. The multifunctional Ca2+ and calmodulin-dependent protein kinase-II (CaMKII) is activated by oxidation of paired methionines. Oxidized CaMKII (ox-CaMKII) is increased in atria from DM patients and causes ryanodine receptor (RyR2) hyperphosphorylation that promotes pathological intracellular Ca2+ release and Ca2+ triggered arrhythmias. We hypothesize that DM increases myocardial ox-CaMKII, RyR2 hyperphosphorylation and AF.

Methods and Results:
C57BL/6J mice with streptozocin-induced type 1 DM had increased AF susceptibility following atrial burst pacing compared with citrate buffer-treated wild-type (WT) controls [70% (14/20) vs. 25% (5/20), p = 0.01]. Ox-CaMKII was increased in atrial tissue from diabetic mice compared to controls, consistent with a role for ox-CaMKII in this model. Diabetic ox-CaMKII resistant knock-in (MM-VV) mice (37.5% (9/24) [p < 0.05]) and diabetic mice with myocardium-restricted transgenic overexpression of methionine sulfoxide reductase A (25% (5/20) [p < 0.05]), which reverses ox-CaMKII, were protected from DM increased AF susceptibility compared to diabetic WT controls. Atrial myocytes from diabetic WT mice demonstrated increased RyR2 mediated Ca2+ spark frequency, triggered action potentials and delayed intracellular [Ca2+] decay compared to controls. Diabetic knock-in mice resistant to CaMKII-mediated RyR2 phosphorylation (S2814A) had decreased AF susceptibility (25% (5/20) [p < 0.05]), compared with diabetic WT mice. All groups of diabetic mice had similar increases in plasma glucose.

Conclusions:
Hyperglycemia increases AF susceptibility and increased ox-CaMKII is associated with increased AF in this diabetic mouse model. Genetic manipulation of an ox-CaMKII pathway can protect against AF susceptibility in DM. These findings suggest that ox-CaMKII is a critical proarrhythmic signal in DM and a potential therapeutic target for AF management in DM patients.
Abstract 46: Study of Symptoms Variation in the Morning and Evening in Postural Orthostatic Tachycardia Syndrome (POTS)
Authors: Ahsan Muhammad M , Thompson Tara , Ashangari Chandralekha , Suleman Amer
Published: June 2021

Background:
Postural Orthostatic Tachycardia Syndrome (POTS) is a form of dysautonomia that is estimated to impact between 1,000,000 and 3,000,000 Americans and millions more around the world. Patients chronically have symptoms that are worse with upright posture and that improve with recumbence. Symptoms often include orthostatic intolerance such as dizziness, fatigue, excessive sweating and many others. The aim of this study is to determine the variation of symptoms early morning after wake up and evening at 4 PM.

Methods:
The Autonomic nervous system questionnaire consisting of eight POTS symptoms palpitations, headaches, dizziness, shortness of breath(SOB), chest pain, weakness, blurred vision and heaviness of feet was handover to the patients at our clinic. 42 POTS patients participated in the study, participated patients had been asked to scale their symptoms early morning after wake up and evening at 4 PM. Symptoms were defined 0 as Never,1 as mild,2 as moderate,3 as severe ,4 as extreme and 5 impairing daily function and living.

Results:
Out of 42 POTS patients 90% are female (38/42, age 31.74±10.67) and 10% are males (4/42, age 30.75±13.20), symptoms were scaled early morning after wake up vs symptoms scaled evening at 4 pm results in mean±SD and Anova P value. Palpitations 1.69±1.32 vs 2.45±1.38 (P =0.01), Dizziness 2.40±1.38 vs 2.67±1.46 (P =0.40), Chest pain 0.93±1.02 vs 1.52±1.27 (P=0.02), SOB 1.36±1.32 vs 1.81±1.40 (P=0.13), Weakness 2.79±1.37 vs 3.12±1.38 (P=0.27), Blurred vision 1.38±1.29 vs 1.48±1.27 (P=0.73), Headaches 1.74±1.36 vs 2.52±1.38 (P =0.01), Heaviness of feet 0.95±1.19 vs 1.43±1.53 (P =0.11).

Conclusion:
Our study results demonstrated that the patients with POTS had significant variation in symptoms Palpitations, Chest pain, Headaches in evenings when compared to early morning after wake up.
Abstract 460: β
1
-Adrenergic Receptor O-glycosylation Regulates Receptor N-terminal Cleavage and Signaling Responses in Cardiomyocytes
Authors: Park Misun , Xiang Yang K , Reddy Gopireddy R , Wallukat Gerd , Steinberg Susan F
Published: June 2021

β1-adrenergic receptors (β1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Recent efforts to structurally characterize β1ARs have focused on the ligand binding sites in transmembrane helices and effector docking sites in the intracellular loops and C-terminus. The notion that the β1AR extracellular N-terminus (which is a substrate for both N- and O-linked glycosylation and a target for proteolytic cleavage) functions as a structural determinant of β1AR activation has never been considered. This study takes advantage of site-directed mutagenesis and expression studies in Chinese Hamster ovary cells that harbor a reversible glycosylation defect to map β1ARs O-glycosylation sites to Ser37/Ser41 and show that O-glycosylation at these sites prevents β1AR N-terminal cleavage. We then used an adenoviral overexpression approach to show that both full-length fully-glycosylated β1ARs (β1AR-FL) and N-terminally truncated glycosylation-defective β1ARs (β1AR-Δ52) increase cAMP accumulation and activate the ERK-MAPK signaling cascade in cardiomyocytes. However, isoproterenol-dependent cAMP accumulation is markedly enhanced, and ERK activation is markedly attenuated, in cardiomyocytes that express β1AR-Δ52, compared with β1AR-FL cultures. The conclusion that β1ARs are stabilized in a conformation that is biased toward the cAMP pathway as a result of N-terminal truncation gains further support from additional studies showing that agonistic β1AR autoantibodies induce a more rapid and robust increase in membrane PKA activity in cells that express glycosylation-defective β1ARs, compared to cells that express full-length glycosylated β1ARs. These results identify a novel role for O-glycosylation as a structural determinant of β1AR responsiveness in the heart.