bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 348: Phorbol 12-myristate 13-acetate Induces Dedifferentiation of Cardiac Myofibroblasts to Fibroblasts and Other Unidentified Type of Cells
Authors: Tran Luu Vy , Phan Sang , Jin Zhuqiu
Published: October 2020

Cardiac fibrosis plays an essential role in cardiac pathogenic processes that occur as a result of myocardial infarction or hypertrophic cardiomyopathy. The differentiation of cardiac fibroblasts to myofibroblasts is considered to be a critical step in the activation and progression of cardiac fibrosis. TGFβ is one of the essential molecules that promote transition of fibroblasts to myofibroblasts. Reversal of formed myofibroblasts to fibroblasts remains incompletely understood. Phorbol 12-Myristate 13-Acetate (PMA) regulates metabolism and functions of multiple cells via PKC activation mostly. To study effects of PMA on differentiation of de novo formed cardiac myofibroblasts, human cardiac fibroblasts were utilized. Human cardiac fibroblasts (HCF) cultured in fibroblast medium (FM)-2 were converted into myofibroblasts in the presence of 2 ng/mL of TGF-β1 for 48 hours. Expression of α-SMA, the biomarker of myofibroblasts, and FSP1, the biomarker of fibroblasts, was detected using Western blot and immunofluorescence. Collagen gel contraction induced by fibroblasts was determined as well. TGF-β1 increased the expression of α-SMA and reduced the expression of FSP1. Distinct cellular morphology changes in the shape and size of HCF were observed after incubation with TGF-β1 for 48 hours. To investigate effect of PMA on dedifferentiation of formed myofibroblasts, these TGF-β1-pretreated cells were divided into four groups for additional 48 hours incubation: PMA groups (10, 50, and 100 ng/mL) or DMSO (vehicle control). Both 50 and 100 ng/mL of PMA reduced the expression of α-SMA but only 100 ng/mL of PMA increased the expression of FSP1. The shape and size of cells changed after treatment with PMA. PMA also reduced TGF-β1-induced collagen gel contraction (P<0.05, compared to DMSO group). These data indicated that PMA can reverse the differentiation of de novo formed human cardiac myofibroblasts induced by TGF-β1 to fibroblasts and other unidentified type of cells. Although the mechanism of dedifferentiation remains to be identified, the novel finding of this study shed light on future development of agents to treat fibrotic diseases.
Abstract 350: Immune Profiling and Causal Antigen Discovery in Mouse and Human Models of Immune Checkpoint Inhibitor-induced Myocarditis
Authors: Zhu Han , Lee Daniel , Sarah Waliany , Galdos Francisco X , D’Addabbo Jessica , Fowler Michael B , Reddy Sunil , Heather Wakelee , Neal Joel W , Witteles Ronald , Maecker Holden T , Davis Mark , NGUYEN Patricia K , Wu Sean M
Published: October 2020

Introduction:
Immune checkpoint inhibitors (ICIs) are novel drugs that activate T cell-mediated anti-tumor response by blocking immune checkpoints such as PD-1 or CTLA-4, leading to improved cancer patient survival. Despite these benefits, ICIs can result in autoimmune side effects including fulminant myocarditis and heart failure. While ICI-induced myocarditis is characterized by myocardial T cell infiltration, the causal mechanisms remain unknown. We hypothesize that ICI-induced myocarditis is caused by cardiac-specific auto-antigens triggering clonal expansion of myocardial CD8+ T-cells, leading to T-cell mediated myocardial damage.

Methods/Results:
We have explored the ICI-induced inflammatory response in a mouse model of myocarditis induced by PD-1 knockout and in patients with ICI-induced myocarditis. PD-1 deficient-mice on a lupus-like autoimmune background (i.e. MRL/Pcd1-/- mice) develop spontaneous fatal myocarditis in 70% of animals by 5 weeks of age, with massive cardiac infiltration of CD8>CD4+ T-cells. Likewise, patients with ICI-induced myocarditis have CD8>CD4+ T-cell infiltrate in the heart. We have performed time-of-flight mass cytometry (CyTOF) to immunophenotype the T-cell subsets in the blood/myocardium of MRL/Pcd1-/- mice and in ICI-myocarditis patients. We have also conducted single cell sequencing of T-cell receptors (TCRs) from the blood +/- myocardial-derived T-cell samples of the mice and patients. Our preliminary results in ICI-myocarditis patients confirmed the previously reported CD8+ T-cell expansion in the blood and myocardium of myocarditis patients compared with healthy control. We are currently identifying candidate cardiac auto-antigen(s) responsible for this disease by performing Grouping Lymphocyte Interactions by Paratope Hotspots (GLIPH).

Conclusion:
Myocarditis is a serious and life-threatening complication of ICI treatment. By understanding the unique immune response present during ICI-induced myocarditis and the responsible cardiac auto-antigen(s) involved, we will pave the way for the development of adjuvant therapies that target these antigens and mitigate their deleterious effects.
Abstract 353: High Density Lipoprotein Regulates Tumor Necrosis Factor Alpha and Jun Kinase Signaling Pathway in Adipocytes
Authors: Zhong Qiaoqing
Published: October 2020

Background:
High-density lipoproteins (HDL) is presumed to exhibit cardiovascular protection, since HDL mediate the reverse transport of cholesterol and exhibit strong anti-inflammatory potential. Previous studies have found that HDL can reduce the level of IL-8 secreted by the inflammatory adipocytes, as well as affect the expression of adiponectin and tumor necrosis factor-alpha (TNF-α) under the inflammatory condition in adipocytes. These studies indicate that HDL is closely related to the inflammatory response of adipose tissue, yet its mechanism has not been fully understood. Secretory leucocyte protease inhibitor (SLPI), a nonglycosylated single-chain polypeptide protein, may play an important role in diet-induced obesity and adipocyte inflammation.

Purpose:
To study the role of SLPI in HDL anti-inflammatory action in the adipocytes, and explore the mechanism of the action.

Methods:
3T3-L1 Adipocytes were induced to differentiation and maturation by culturing. Acute inflammation in adipocytes was induced by LPS (100 ng/ml, 6h), and then the adipocytes were pretreated with HDL and/or SLPI-siRNA. The protein and mRNA levels of SLPI, TNF-α and JNK were examined by Western blotting and qRT-PCR assay, respectively. In addition, the levels of inflammatory factors were measured by ELISA.

Results:
HDL up-regulated SLPI expression, down-regulated TNF-α expression, and inhibited the secretion of inflammatory factors such as IL-1, IL-6, IL-8 and MCP-1 in 3T3-L1 Adipocytes pre-treated by LPS. Silencing of SLPI by its siRNA effectively abolished the anti-inflammatory effect of HDL. JNK inhibitor (SP600125) partially restored the anti-inflammatory effects of HDL decreased by SLPI knockdown and decreased the secretion of inflammatory factors.

Conclusions:
These data suggest that HDL up-regulate the expression of SLPI and inhibit the inflammation in 3T3-L1 Adipocytes pre-treated by LPS. The anti-inflammatory effect of HDL, including inhibition of the secretion of inflammatory factors via TNF-α/JNK signaling pathway, probably viat up-regulating the SLPI expression.

Key words:
high-density lipoprotein, Inflammation, Adipocytes
Abstract 354: The Role of the Liver Heart Axis During Cardiac Remodeling
Authors: Usui Soichiro , Takashima Shin-ichiro , Sakata Kenji , Kawashiri Masa-aki , Takamura Masayuki
Published: October 2020

Background:
Hepatokine selenoprotein P (SeP) contributes to insulin resistance and hyperglycemia in patients with type 2 diabetes. Although clinical studies suggest the insulin resistance is an independent risk factor of heart failure and inhibition of SeP protects the heart from ischemia reperfusion injury, the role of SeP in pathogenesis of chronic heart failure is not well understood.

Objective:
We examined the role of SeP in the regulation of cardiac remodeling in response to pressure overload.

Methods and Results:
We measured serum SeP levels in 22 patients for heart failure with reduced ejection fraction (HFrEF; LVEF<50%) and 22 normal subjects. Serum levels of SeP were significantly elevated in patients with HFrEF compared to in normal subjects (3.55 ± 0.43 vs 2.98 ± 0.43, p<0.01). To examine the role of SeP in cardiac remodeling, SeP knockout (KO) and wild-type (WT) mice were subjected to pressure overload (transverse aortic constriction (TAC)) for 2 weeks. The mortality rate following TAC was significantly decreased in SeP KO mice compared to WT mice (22.5 % in KO mice (n=40) vs 52.3 % in WT mice (n=39) p<0.01). LV weight/tibial length (TL) was significantly smaller in SeP KO mice than in WT mice (6.75 ± 0.24 vs 8.33 ± 0.32, p<0.01). Lung weight/TL was significantly smaller in SeP KO than in WT mice (10.46 ± 0.44 vs 16.38 ± 1.12, p<0.05). Interestingly, hepatic expression of SeP in WT was significantly increased by TAC. To determine whether hepatic overexpression of SeP affects TAC-induced cardiac hypertrophy, a hydrodynamic injection method was used to generate mice that overexpress SeP mRNA in the liver. Hepatic overexpression of SeP in SeP KO mice lead to a significant increase in LV weight/TL and Lung weight/TL after TAC compared to that in other SeP KO mice.

Conclusions:
These results suggest that serum levels of SeP were elevated in patients with heart failure with reduced ejection fraction and cardiac pressure overload induced hepatic expression of SeP in mice model. Gene deletion of SeP attenuated cardiac hypertrophy and dysfunction in response to pressure overload in mice. SeP possibly plays a pivotal role in promoting cardiac remodeling through the liver-heart axis.
Abstract 358: Hypoxia and Hyperglycemia Induces M
6
A Mrna Methylation in Major Heart Cells
Authors: Kumari Rajesh , Ranjan Prabhat , Suleiman Zainab , Li Jing , Verma Suresh K
Published: October 2020

Background:
According to American heart association, over 70 % diabetic patients die from heart and stroke related diseases. The term “diabetic heart diseases” includes coronary heart disease, heart failure and diabetic cardiomyopathy in diabetic patients. Epigenetic and epitranscriptomic modifications play critical roles in progress of diabetic heart disease. Recent evidences indicated that m
6
A methylation involved in ischemic cardiomyopathy. But, the role of m
6
A mRNA methylation in cardiovascular diseases along with diabetic co-morbidity factors has not been studied in details. Thus, here we hypothesize that alterations in m
6
A mRNA methylation under hypoxic and hyperglycemic conditions contributes to severity of ischemic heart disease.

Method and Results:
To address our hypothesis, we have determined the levels of m
6
A mRNA methylation in NRVM, NRVF and HUVEC, HMVE and mouse primary endothelial cells under hypoxic and hyperglycemic conditions. We also examined the m
6
A levels in mice hearts post 5 days of MI. m
6
A mRNA methylation was significantly upregulated both in human and mouse ischemic hearts. Furthermore, hypoxia and hyperglycemia significantly induced m
6
A methylation in neonatal rat cardiomyocytes, fibroblasts, and mouse primary endothelial cells (isolated from WT and db/db mice). Next, we measured the methylation machinery both at RNA and protein levels. Interestingly, in corroboration with our methylation data, the expression of both m
6
A writers (Mettl3 and WTAP) and Readers (YTHDF2) was significantly increased. To determine the target transcripts which were highly methylated post-ischemia, we performed deep sequencing of methylated RNA after their immunoprecipitation using MeRIP-sequencing protocol. Our MeRIP-seq data has suggested a differentially m
6
A methylated targets both in-vitro and in-vivo ischemic sample.

Conclusion:
Over all, for the first time our data showed that hypoxia and hyperglycemia alters m
6
A mRNA methylation which may contribute to enhance the severity of cardiovascular diseases under hyperglycemic conditions. Further understanding of the mechanisms, may present a novel approach to potentially regulate m
6
A methylation, which may help in preventing/reducing heart failure in diabetic patients.
Abstract 359: Cardiac-specific Deletion of Prohibitin-1 Leads to Fetal Demise, Gross Mitochondrial Abnormalities and Heart Failure in Adolescent Mice
Authors: Huo Ran , Zimmerman Kathy , Weiss Robert M , Bowdler Noelle C , Anderson Ethan J
Published: October 2020

Prohibitins (PHB1, 2) are lipid raft-associated proteins which form a multimeric, alternating ring-like structure in cellular membranes, and are particularly abundant in mitochondria and plasma membranes, but have also been found in nuclei. These pleiotropic proteins are associated with numerous signaling pathways responsible for cell differentiation, growth, and metabolism. In mitochondria, PHBs are thought to act as scaffold-like chaperone proteins responsible for stabilizing enzymes responsible for electron transport and oxidative phosphorylation (OXPHOS), minimizing reactive oxygen species (ROS), and coordinating fission/fusion. However, the extent to which PHB1 regulates mitochondria and electromechanical function of the heart, is not known. We established a cardiac-specific PHB1-deficient mouse strain by crossing floxxed
phb1
mice with mice expressing Cre recombinase under control of myosin heavy chain (MHC) promoter. The vast majority of cardiac-specific PHB1-deficient mice (cPHB1ko) died in utero during pregnancy (E12-E15). Uterine sonography of the expired cPHB1ko embryos showed significantly decreased crown-rump length and increased placenta thickness with large amount of fluids, restricted uterine artery flow and detachment between amnion and chorion compared with normal embryos (P < 0.001). The few that were born develop severe dilated cardiomyopathy, resulting in mortality by 8-9 weeks of age. Heart weight/body weight ratio is ~2-fold greater in the KO mice vs. WT. Echocardiography reveals severely enlarged right atria and ventricles in cPHB1ko mice, corresponding to significantly decreased ejection faction (EF), increased end diastolic volume (EDV) and end systolic volume (ESV) compared to wild-type littermates (P < 0.05). In mitochondria, cPHB1ko mice have derangements in OXPHOS as evidenced by decreased pyruvate and palmitoylcarnitine-supported respiration and ATP production. Moreover, both the rate of calcium uptake (P = 0.05) and the total calcium retention capacity (P < 0.01) were diminished in the KO mice. These preliminary findings implicate PHB1 as a critical regulator of myocardial development and function, due in part to its role in maintaining OXPHOS and controlling metabolism.
Abstract 362: Role of Dysregulated Exosomal MiRNAs in Functional Impairment of Cardiac Endothelial Cells
Authors: Ranjan Prabhat , Kumari Rajesh , Pal Harish , Krishnamurthy Prasanna , Qin Gangjian , Kishore Raj , Verma Suresh K
Published: October 2020

Objective:
Endothelial cells (ECs) play critical role to maintain the normal heart function. It is shown that fibroblast-derived exosomes have the ability to enhance cardiac myocytes hypertrophy in pressure-overloaded myocardium. However, their effect on endothelial cell function has not been studied. Others and we have previously shown that stress-induced chronic inflammation induces cardiac fibroblasts and mediates endothelial cells dysfunction. Here we hypothesized that activated cardiac fibroblasts-derived exosomes (FB-Exo) mediates cardiac ECs dysfunction and leads for cardiac pathology and restoring the altered FB-Exo contents will improve endothelial cells function and biology.

Methods:
We cultured mouse primary endothelial cells in EC growth media. Cells were treated with fibroblasts-derived-Exo. Exosomes were isolated from fibroblast condition media by ultracentrifugation and characterized by nanosight & electron microscopy.

Results:
Fibroblasts were significantly activated by TGFβ treatment as shown by qPCR and western blot (smad2/3, p-Smad2/3, p38, p-p38) data. Endothelial cells dysfunction as shown by Matrigel assay, real-time Q-PCR and western data (eNOS and Hif1α) data was observed in TGFβ-FB-Exo treatment endothelial cell. MTT, TUNEL and migration assay also followed the same trend as TGFβ-FB-Exo treatment significantly induced endothelial cell death and inhibits its proliferation and migration. Furthermore, microRNA array and PCR analysis revealed dysregulation of miR-132-3p, miR-2001-3p and miR-125b-5p in TGFβ-FB-Exo treated endothelial cells.

Conclusions:
Taken together, this study demonstrates that TGFβ treated fibroblasts-derived exosomes are enriched in pro-fibrotic factors and can lead to endothelial dysfunction and promotes cardiac fibrosis in PO myocardium. In future study, we will modulate the target miRs in fibroblasts to see whether it rescue reparative function of endothelial cell and inhibits cardiac fibrosis in failing heart.
Abstract 365: Ponatinib Triggers Cardiac Inflammation by STAT-3 Dependent Immune Checkpoint Blockade
Authors: Singh Anand P , Sultan Tousif , Umbarkar Prachi , Zhang Qinkun , Lal Hind
Published: October 2020

Background:
Ponatinib is a potent anticancer tyrosine kinase inhibitor (TKI) with considerable cardiotoxicity. The manifestation of cardiovascular adverse events, including fatal myocardial infarction and congestive heart failure, has hampered its clinical use. Therefore, a better understanding of the mechanism by which ponatinib exerts cardiotoxicity is urgently warranted to efficiently counteract treatment-related adversities.

Methods:
Wild type C57BL/6, comorbidity mouse model ApoE
-/-
,
and pressure overload (PO) mouse model were used to investigate the cardiotoxic mechanism of ponatinib. Echocardiography was performed to assess cardiac function. Flow cytometry analysis was performed to assess the dynamics of inflammation.

Results:
We observed that high-fat diet (HFD) fed ApoE
-/-
mice develop cardiac dysfunction within 2 weeks of ponatinib treatment. An unbiased RNA-Seq analysis revealed significant upregulation of inflammatory genes (CCR1, CCR5, CCL6, CCL8, CCL9, CXCL4) in ponatinib treated hearts. Since ApoE
-/-
background, and HFD are known confounder of inflammation signal, we validated this observation in naïve C57BL/6 mice. Despite the lack of cardiac dysfunction in ponatinib treated naïve C57BL/6 mice, comprehensive immune profiling depicted upregulation of myocardial inflammation as evident by infiltrated immune cells (CD45
+
TNFα
+
, CD45
+
CD11b
+
F4/80
+
Ly6C
+
CCR2
+
, CD45
+
Gal1
+
). Interestingly, we also demonstrated the downregulation of immune checkpoints over T cells (TCRαβ
+
CTLA4
+
, TCRαβ
+
PD1
+
) in ponatinib treated hearts. Next, in the PO mouse model, ponatinib treated mice showed significant cardiac dysfunction with myocardial inflammation as reflected by increased frequencies of inflammatory parameters (TNFα
+
, IL1β
+
, IL6
+
, MCP1
+
,
CXCL9
+
). Mechanistically, we demonstrated that ponatinib potentially suppresses PD-L1 expression over cardiomyocytes via inhibition of STAT3, subsequently leading to immune cells mediated myocardial inflammation.

Conclusions:
These findings uncover a novel mechanism of ponatinib induced cardiac inflammation leading to adverse cardiac function. It also suggests that strategies to attenuate inflammation may be an effective therapy to prevent ponatinib induced cardiac adverse events.
Abstract 368: Mechanisms of Arrhythmogenic Substrates in Lipotoxic Heart
Authors: Martinez -Mateu Laura , Zhang Xusheng , Leduc Charles , Leibel Rudolph , Saiz Javier , Aromolaran Ademuyiwa
Published: October 2020

Obesity is associated with dangerous pathologies including insulin resistance, hyperglycemia, and diabetes all of which are present as independent risks to developing fatal arrhythmias, which lead to sudden cardiac death. Cardiac lipotoxicity is a common mechanism that links these pathologies to cardiac dysfunction. The molecular mechanisms of cardiac lipotoxicity in obese heart are unknown. We investigated the effects of high-fat diet (HFD)-induced lipotoxicity on atrial electrical remodeling. Electrophysiology and rapid atrial pacing (RAP) were used to evaluate the effects of cardiac lipotoxicity in obese guinea pig hearts that show no signs of hyperglycemia or inflammation. HFD atria were associated with increased voltage-dependent potassium (I
K
) and decreased L-type calcium current (I
Ca,L
) densities, spontaneous beats, and increased vulnerability to atrial tachycardia with pacing. Further we see a marked reduction in I
Kur
and increased I
K1
phenotype only after RAP. Human cardiac computer simulation studies underscore the translational relevance, as results were identical to data in lipotoxic guinea pig model. The data are the first to show that I
K
and I
Ca,L

underlie initiation of atrial arrhythmogenesis, while IK
ur
and IK
1
may act to sustain the arrhythmia. RNA sequencing assay in lipotoxic myocytes further revealed upregulation of PI3K and/or downregulation of AMPK as prime candidates for modulation of atrial ionic currents. The data provide a unique mechanism-based insight for targeted treatment options in patients.
Abstract 371: Cardiac Fibrosis is Associated With Increased Gut Barrier Leakage and Circulating Peptidoglycan in Type 2 Diabetic Mice With Ace2 Deficiency
Authors: Prasad Ram , Ranjan Prabhat , Verma Suresh K , Grant Maria
Published: October 2020

Purpose:
Cardiac fibrosis is linked with hyperglycemia and increases the risk of cardiovascular complications and heart failure. Despite several advancements, the molecular mechanisms responsible for cardiac fibrosis in diabetes remain unclear. The renin-angiotensin system (RAS) is activated and plays an essential role in diabetes-induced cardiac dysfunctions through activation of Ang II. However, angiotensin converting enzyme 2 (ACE2), the protective arm of RAS, opposes the classical RAS functions mediated by ACE. Previously, we have reported that ACE2 deficient exacerbates hyperglycemia-induced gut dysbiosis, increased gut barrier permeability, and facilities translocation of gut microbial peptides (GMPs) into system circulation in type 1 diabetic mice. Here, we sought to determine whether dysbiosis induced by type 2 diabetes and ACE2 deficiency contributes to cardiac fibrosis.

Methods:
Toll-like receptor- (TLR-2) associated inflammatory signaling and cardiac fibrosis were studied in WT, db/db, and ACE2
-/y
-db/db mice with 4-months of diabetes. The levels of peptidoglycan (PGN), a TLR-2 ligand, were measured by ELISA assay. The markers of gut barriers and inflammatory cytokines were assessed by immunohistochemistry and qRT-PCR. To measure cardiac fibrosis, Masson’s trichrome staining was used to stain collagen fibers.

Results:
ACE2
-/y
-db/db mice exhibited increased myocardial fibrosis as compared with db/db and WT mice. In the myocardium, TLR-2 expression (4 folds, p<0.005) and pro-inflammatory cytokines IL-1β (p<0.001), IL-6 (p<0.0001), and TNF-α (p<0.01 were significantly up-regulated in ACE2
-/y
-db/db mice compared with db/db and WT mice. Increased gut barrier permeability as detected by a reduction in expression of tight junction molecules (ZO-1, p120-catenin, and VE-cadherin) and a 2- fold increase in circulating PGN was observed in ACE2
-/y
-db/db mice when compared to db/db.

Conclusions:
These data suggest that PGN and other circulating gut microbial particles may have a deleterious impact on cardiac health by promoting inflammation-mediated fibrosis. In addition to controlling hyperglycemia, strategies aimed at restoring gut barrier integrity may prevent diabetic cardiovascular complications.
Abstract 372: Misoprostol Attenuates Hypoxia-induced Cardiomyocyte Proliferation Through Bnip3 and Perinuclear Calcium Signaling
Authors: Martens Matthew D , Field Jared T , Seshadri Nivedita , Day Chelsea , Doucette Christine , West Adrian R , Ivanco Tammy L , Gordon Joseph W
Published: October 2020

Systemic hypoxia resulting from preterm birth, altered lung development, and cyanotic congenital heart disease is known to impede the regulatory and developmental pathways in the neonatal heart. While the molecular mechanisms are still unknown, hypoxia induces aberrant cardiomyocyte proliferation, which may be initially adaptive, but can ultimately program the heart to fail in early life. Recent evidence suggests that the prostaglandin E1 analogue, misoprostol, is cytoprotective in the hypoxia-exposed neonatal heart by impacting alternative splicing of the Bcl-2 family member Bnip3, resulting in the generation of a variant lacking the third exon (Bnip3
Δ
Exon3 or small Nip; sNip). Using a rodent model of neonatal hypoxia, in combination with rat primary neonatal cardiomyocytes (PVNC’s) and H9c2 cells, we sought to determine if misoprostol can prevent cardiomyocyte proliferation and what the key molecular mechanisms might be in this pathway. In PVNC’s, exposure to 10% oxygen induced myocyte proliferation concurrent with molecular markers of cell-cycle progression, such as Cyclin-D1, which were prevented by misoprostol treatment. Furthermore, we describe a critical role for sNip in opposing cardiomyocyte proliferation through several mechanisms, including reduced expression of the proliferative MEF2C-myocardin-BMP10 pathway, promoting nuclear calcium accumulation leading to NFATc3 activation, and increased expression of the cardiac maturation factor BMP2. Intriguingly, misoprostol and sNip inhibited a hypoxia-induced glycolytic flux, which directly influenced myocyte proliferation. These observations were further supported by knockdown studies, where hypoxia-induced cardiomyocyte proliferation is restored in misoprostol-treated cells by an siRNA targeting sNip. Finally, in postnatal day (PND)-10 rat pups exposed to hypoxia, we observed histological evidence of increased nuclei number and increased PPH3 staining, without fibrosis which were completely attenuated by misoprostol treatment. Collectively, this data demonstrates how neonatal cardiomyocyte proliferation can be pharmacologically modulated by misoprostol treatment, which may have important implications for both neonatal and regenerative medicine.
Abstract 381: Mcur1 is a Potential Target to Modulate Cardiac Contractility and Alleviate Cardiac Function During Heart Failure
Authors: Roy Rajika , Shanmughapriya Santhanam , Zhang Xueqian , Song Jianliang , Tomar Dhanendra , M. Schumacher Sarah , Gao Erhe , Wang Hong , Y. Cheung Joseph , J. Koch Walter , Rajan Sudarsan
Published: October 2020

Cardiac contractility is regulated by the intracellular Ca
2+
concentration fluxes which are actively regulated by multiple channels and transporters. Ca
2+
uptake into the mitochondrial matrix is precisely controlled by the highly Ca
2+
selective channel, Mitochondrial Calcium Uniporter (MCU). Earlier studies on the cardiac-specific acute MCU knockout and a transgenic dominant-negative MCU mice have demonstrated that mitochondrial Ca
2+
(
m
Ca
2+
) signaling is necessary for cardiac ‘‘fight-or-flight’’ contractile response, however, the role of
m
Ca
2+
buffering to shape global cytosolic Ca
2+
levels and affect E-C coupling, particularly the Ca
2+
transient, on a beat-to-beat basis still remains to be solved. Our earlier studies have demonstrated that loss of MCU Regulator 1 (MCUR1) in cardiomyocytes results in the impaired
m
Ca
2+
uptake. We have now employed the cardiac-specific MCUR1 knockout mouse to dissect the precise role of MCU in regulating cytosolic Ca
2+
transients associated with excitation-contraction (E-C) coupling and cardiac function. Results from our studies including the in vivo analyses of cardiac physiology during normal and pressure-overloaded mouse models and in vitro experiments including single-cell cardiac contractility, calcium transients, and electrophysiology measurements demonstrate that MCUR1/MCU regulated
m
Ca
2+
buffering in cardiomyocytes, although insignificant under basal condition, becomes critical in stress induced conditions and actively participates in regulating the
c
Ca
2+
transients. Also, the ablation of MCUR1 in cardiomyocytes during stress conditions prevents
m
Ca
2+
overload and subsequent mROS overproduction. Our data indicate that MCUR1 ablation offers protection against pressure-overload cardiac hypertrophy. In summary, our results provide critical insights into the mechanisms by which the MCU channel contributes in regulating the contractile function of the cardiomyocytes and the role of
m
Ca
2+
in the development and progression of heart failure.
Abstract 391: Mir-A-3p Targets Atf3 to Reduce Calcium Deposition in Vascular Smooth Muscle Cells
Authors: CHOE Nakwon , Kwon Duk-hwa , Ryu Juhee , SHIN SERA , Joung Hosouk , Ahn Youngkeun , Kim Young-Kook , Kook Hyun
Published: October 2020

Vascular calcification, the ectopic deposition of calcium in blood vessels, develops in association with various metabolic diseases and atherosclerosis and is an independent predictor for morbidity and mortality of these diseases. Here we report that reduction of microRNA-A-3p (miR-A-3p) causes an increase in ATF3, activating transcription factor 3, a novel osteogenic transcription factor, in vascular smooth muscle cells. Both miRNA and mRNA microarrays were performed with rat vascular smooth muscle cells and reciprocally regulated pairs of miRNA and mRNA were selected after bioinformatic analysis. Inorganic phosphate significantly reduced the expression of miR-A-3p in A10 cells. The transcript level was also reduced in vitamin D3-administered mouse aortas. miR-A-3p mimic reduced calcium deposition, whereas miR-A-3p inhibitor increased it. The Atf3 mRNA level was upregulated in cellular vascular calcification model, and miR-A-3p reduced the Atf3 mRNA and protein levels. Transfection with Atf3 could recover the miR-A-3p-induced reduction of calcium deposition. Our results suggest that reduction of miR-A-3p may contribute to the development of vascular calcification by de-repression of ATF3
Abstract 393: S-nitrosylation of Atg7 by Thioredoxin-1 (trx1) Protects the Heart Against Myocardial Ischemia via Autophagy Regulation
Authors: Nagarajan Narayani , Oka Shinichi , NAH Gihoon , Zhai Peiyong , Mizushima Wataru , Mukai Risa , Xu Xiaoyong , Huang Chun-yang , Wu Changgong , Liu Tong , Li Hong , Sadoshima Junichi
Published: October 2020

Thioredoxin 1 (Trx1) is an oxidoreductase that reduces proteins with disulfide bonds. Trx1 also functions as a transnitrosylase, but this occurs only when Trx1 is oxidized at Cys32 and Cys35. In cultured cardiomyocytes (CMs), glucose deprivation (GD) induces oxidation of Trx1 and Trx1 is transnitrosylated. Thus, we hypothesized that Trx1 promotes GD-induced autophagy through its function as a transnitrosylase rather than as an oxidoreductase. GD-induced autophagy, evaluated by counting GFP-LC3 puncta, was inhibited in the presence of the transnitrosylation-defective Trx1C73S mutant (GFP-LC3 puncta per cell under GD; control: 38; Trx1 knockdown: 2*; Trx1C73S: 17*; p<0.05 vs. control), suggesting that Cys73 in Trx1 plays an important role in mediating GD-induced autophagy. Mass spectrometric analyses and biochemical assays showed that Atg7, an essential autophagy regulator, is a Trx1 target and that Trx1 binds to Atg7 via Cys454 and Cys458 in Atg7, thereby transnitrosylating Atg7 at Cys294 and Cys402. Trx1C73S knock-in (Trx1C73S-KI) promoted coronary ligation (CL)-induced myocardial infarction (MI) (MI size/area at risk (AAR) (%); Wild type (WT): 21; Trx1C73S-KI: 42*; p<0.05 vs. WT), in association with reduced S-nitrosylation of Atg7. To test the role of S-nitrosylation of Atg7 in mediating autophagy, we transduced an S-nitrosylation defective Atg7 mutant (Atg7CC294/402SS) into adult cardiomyocytes isolated from cardiac-specific Atg7 knockout mice. Compared to intact Atg7, Atg7CC294/402SS was less able to induce autophagy, as evidenced by reduced LC-3II formation (relative LC3-II; intact Atg7: 1.0; Atg7CC294/402SS: 0.81*; p<0.05). Atg7C402S, but not Atg7C294S, knock-in exacerbated CL-induced MI (MI size/AAR (%); WT: 32; Atg7C402-KI: 42*; p<0.05 vs. WT). These results suggest that Trx1 protects the heart against MI by mediating autophagy via S-nitrosylation of Atg7 at Cys402.
Abstract 396: 3d-Engineered Aligned Co-axial Nanofibrous Cardiac Patch for Drug Screening and Cardiac Repair Applications
Authors: Khan Mahmood , Sridharan Divya , Kumar Naresh , Palaniappan Arunumar , Dougherty Julie A , Isai Dona G , Czirok Andras , Powell Heather
Published: October 2020

Introduction:
Recent studies have demonstrated the great potential of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) for testing the efficacy of various cardiac drugs. Additionally, studies have shown that the hiPSC-CMs grown in a 3D environment express better physiological characteristics than 2D cultures. The convergence of polymeric cardiac patch technology with hiPSC-CMs has opened up innovative ways for generating biomimetic 3D cardiac tissues.

Hypothesis:
The central hypothesis of this study was to develop a 3D cardiac tissue model for pharmacological testing of various cardiac drugs on a 3D nanofibrous aligned co-axial cardiac patch.

Methods:
A co-axial (Co-A) PCL-gelatin aligned nanofibrous patch was fabricated using the electrospinning technique and its mechanical properties were assessed using Universal Test Machine. Then, the hiPSC-CMs were cultured on this Co-A patch for 2 weeks and the LDH assay was performed to determine the cell viability. The functionality of the cardiac patch was determined by an assessment of calcium cycling in hiPSC-CMs. Further, particle image velocimetry (PIV) and microelectrode array (MEA) was used to evaluate the physiological functionality of the cardiac patch in response to various cardiac drugs.

Results:
Our studies showed that the mean diameter and thickness of aligned Co-A nanofibrous patch was 578±184 nm and 115±11 μm respectively, while its tensile strength was 0.780 ± 0.098 MPa. Further, confocal imaging confirmed the core-shell structure of the Co-A patches with a core diameter of 2.21 ± 0.50 μm. Additionally, The hiPSC-CMs cultured on these aligned Co-A patches showed an aligned morphology and expressed Troponin-T, GATA4, α-sarcomeric actinin, and connexin-43. The hiPSC-CMs seeded on a 3D scaffold showed efficient calcium cycling properties, which were similar to the hiPSC-CMs cultured in 2D scaffold. Furthermore, PIV and MEA analysis showed that hiPSC-CMs cultured in 2D and 3D showed a similar response to various cardiac drugs, isoproterenol, verapamil and E4031.

Conclusions:
Overall, this study demonstrated a successful fabrication of aligned Co-A nanofibrous cardiac patch and its evaluation as a 3D cardiac tissue model in-vitro, which could be applied towards drug screening, toxicity studies and cardiac repair applications for ischemic heart disease.
Abstract 400: Tip60 Depletion in Neonatal Cardiomyocytes Reduces the DNA Damage Response and Increases Cell-cycle Activation
Authors: Wang Xinrui , Lupton Carri , Lauth Amelia , Wan Tina C , Patterson Michaela , Auchampach John A , Lough John W
Published: October 2020

Tip60 (
T
at
i
nteractive
p
rotein, 60 kD) is a tumor suppressor encoded by the
Kat5
gene that functions as a pan-acetylase. Among its targets, Atm (
a
taxia-
t
elangiectasia
m
utated) undergoes auto-phosphorylation (pATM) when acetylated by Tip60 to induce the DNA damage response (DDR). Because pAtm-induced activation of the DDR is known to cause proliferative senescence in neonatal cardiomyocytes (CMs), we hypothesize that Tip60 is one of the upstream components in this pathway. Using mice containing floxed
Kat5
alleles and a tamoxifen-inducible Cre-recombinase transgene driven by

Myh6 (Kat5
flox/flox;Myh6-merCremer

), we assessed the effect of depleting Tip60 from neonatal CMs via a single injection of tamoxifen on postnatal day 0 (P0). Immunofluorescent microscopy revealed reduced pAtm-positive CMs at three postnatal stages (P7, P12, P39), accompanied by activation of the cell-cycle as indicated by significantly increased percentages of 5’-bromodeoxyuridine (BrdU)-, phosphohistone H3 (pH3)- and Ki67-positive CMs. In addition to cell-cycle activation, Tip60 depletion promoted nuclear division followed by cytokinesis, as indicated by increased percentage of mononuclear diploid CMs at P12. Accordingly, qPCR revealed that Tip60 depletion increased expression of mRNAs encoding the fetal markers
Nppa, Nppb, Myh7
, and
Acta1
at P39, which in the absence of CM hypertrophy (assessed by WGA staining) suggested maintenance of a proliferation-competent state. These effects occurred concomitant with depressed levels of mRNAs encoding cell-cycle inhibitors (
Meis1, p27
) and a trend toward increased levels of G
2
-phase cell-cycle activators (
cyclins A2, B1
;
Cdk1
) in Tip60-depleted hearts. These findings suggest that depletion of Tip60 at birth inhibits the DDR and delays replicative senescence in neonatal CMs.
Abstract 401: Head to Heart: Mental Health as a Precursor for Heart Disease in San Joaquin Muslim Population
Authors: Siddiqui Maha
Published: October 2020

Introduction:
Islamophobia affects the mental health of the large San Joaquin (SJ) Muslim population leading to psychological distress and mental health concerns which can further lead to High Blood Pressure (HBP) and cardiovascular disease (CVD). SJ has a growing rate of Muslim immigrants who may be at risk for psychological distress and CVD.

Objectives:
Trainees conducted Kessler psychological distress tests, HBP and CVD screenings at mosques to reduce the risk of CVD and spread awareness about mental health, CVD and its implications in the Muslim population due to environmental racism in the most underserved county for health care, SJ.

Methods:
I trained medically ambitious low-income minority female high school students to conduct weekly Kessler psychological distress tests, blood pressure screenings and serve as a health technology coach. Each student monitored 2 hypertensive and psychological distressed patients of 40 hypertensive, psychological distressed, at risk for CVD and low-income Muslim residents. Kessler psychological distress tests and CVD screenings were conducted at all SJ mosques to spread awareness about mental health and heart health and track psychological distress and HBP in the Muslim community.

Findings:

-About 58% (304 of 527) of SJ Muslim population are at risk for CVD

-About 42% (221 of 527) of SJ Muslim population are Stage 1 hypertensive

-About 19% (98 of 527) of SJ Muslim population are Stage 2 hypertensive

-About 61% (319 of 527) of SJ Muslim population are hypertensive and psycologically distressed

Direct Outcomes:

-Youth learned the value of empathy, multiculturalism and service to disadvantaged communities through the program and volunteering

-Female empowerment and program students are determined to pursue medicine and address community concerns

-Improved patient provider trust

-Development of self advocacy for health in the Muslim communityIncreased awareness on CVD, HBP, and mental health among the Muslim communityIncreased participation of youth and females in an otherwise adult and male dominant environments (i.e. mosques)

-Awareness, education among Muslim community for better heart and mental health

-Better understanding in how to manage and seek referral for mental health concerns
Abstract 402: Human CD271-positive Adipose Derived Stem Cells are the Angiogenic Subset Decreased by Donor Insulin Resistance
Authors: Inoue Oto , Usui Soichiro , Yamaguchi Kosei , Takeda Yusuke , Goten Chiaki , Nomura Ayano , Takashima Shinichiro , Sakata Kenji , Kawashiri Masa-aki , Takamura Masayuki
Published: October 2020

Introduction:
Type 2 diabetes mellitus (T2DM) is an important risk factor for cardiovascular diseases (CVDs). Recently, cell therapy using adipose-derived stem cells (ADSCs) has emerged as an attractive therapy for severe CVDs because of their angiogenic potentials. However, whether and how T2DM would impair human ADSC angiogenic capacity is still uncertain. We previously reported that CD31
-
CD34
+
CD271
+
ADSCs (CD271
+
ADSCs) were specifically decreased in adipose tissue of T2DM patients. Therefore, we aimed to investigate the angiogenic capacity of CD271
+
ADSCs. Furthermore, we evaluate which patients’ parameters regard as T2DM would decrease the amount of CD271
+
ADSCs.

Methods and Results:
Human CD45
-
CD34
+
CD31
-
ADSCs were obtained from subcutaneous adipose tissue of healthy donors, separated into CD271
+
and CD271
-
subsets by FACS, and cultured. Both subsets of ADSCs were assessed gene expression profile by microarray. Microarray analysis and validation PCR elucidated that PI3K/Akt/mTOR pathway was significantly up-regulated in CD271
+
ADSCs compared to in CD271
-
ADSCs. (

p

< 0.05). Then, we compared in vivo angiogenic capacity in xenograft experiments of nude mice subjected to hindlimb ischemia. Angiogenesis was evaluated histologically using perfused lectin (capillary density) at day 14. Cell therapy using CD271
+
ADSCs demonstrated about 3-fold more lectin
+
capillaries compared to CD271
-
ADSCs or PBS injection (

p

< 0.005,

n

= 5 / group). Next, we established cultured ADSCs obtained from CD271 knock-out mice (KO-ADSCs) and compared their angiogenic capacity with those from WT mice. Consistently, KO-ADSCs demonstrated impaired in vivo angiogenic capacity (

p

< 0.005,

n

= 5 / group). Finally, we collected 23 samples of adipose tissue obtained from CVD patients and evaluated the frequency of CD271
+
ADSCs in CD45
-
CD34
+
CD31
-
ADSCs. Among studied parameters, HOMA-IR, an index of insulin resistance, was negatively correlated with the frequency of CD271+ ADSCs (

r

= -0.64,

p

< 0.005).

Conclusions:
Human CD271
+
ADSCs demonstrated enhanced in vivo angiogenic capacity with higher mTOR expression. Donor insulin resistance might decrease this regenerative subset of ADSCs. These findings would be critical for development and improvement of ADSC therapy.
Abstract 404: Acute Knock-down of Cardiac Peroxisome Proliferator Activated Receptor α Does Not Impair Cardiac Function
Authors: Fillmore Natasha , Sun Junhui , Springer Danielle , Murphy Elizabeth
Published: October 2020

Alterations in glucose and fatty acid metabolism are believed to contribute to the development of heart failure. Peroxisome Proliferator Activated Receptor α (PPARα) is a transcription factor that regulates fatty acid metabolism and is frequently reported to be reduced in heart failure. However, it is controversial whether this decline in PPARα mediates the development of cardiac hypertrophy and heart failure. To improve our understanding of the role of cardiac PPARα we generated a tamoxifen inducible cardiac-specific PPARα knockout mouse (cPPAR
-/-
). Control (Mer-Cre-Mer and Flox
-/-
) mice and cPPAR
-/-
(Mer-Cre-Mer and Flox
+/+
) mice were treated with tamoxifen at ~2.5 months and were studied 5 weeks after treatment. We verified loss of cardiac PPARα using western blot. cPPAR
-/-
mice appear healthy with normal body weight gain and survival. To examine the impact of cardiac deletion of PPARα on cardiac function we performed echocardiography on control and cPPAR
-/-
. There was no reduction in systolic function between control and cPPAR
-/-
mice. Ejection fraction (Control, 56.3±0.9; cPPAR
-/-
, 59.7±0.1) and fractional shortening (Control, 29.1±0.5; cPPAR
-/-
, 31.5±0.1) were similar in cPPAR
-/-
compared to control hearts. Interestingly however, baseline heart rate was significantly lower in cPPAR
-/-
versus control mice (Control, 531.3±18.3; cPPAR
-/-
, 459.8±2.9 bpm). In addition to having normal cardiac function, heart weights were similar between control and cPPAR
-/-
mice. Overall, these data indicate that an acute reduction in myocardial PPARα per se does not cause cardiac dysfunction. However these data do not exclude the possibility that loss of PPARα could drive cardiac pathology in the context of other signals.
Abstract 407: Beta-blockers Reverse Beta Adrenergic Receptors Desensitization Under Hypoxia
Authors: Sun Yu , Gupta Manveen K , Stenson Kate , Prasad Sathyamangla
Published: October 2020

Hypoxia to heart or brain is a primary cause of heart failure or stroke. Studies have shown that hypoxia increases the beta-adrenergic receptors (βARs) phosphorylation and dysfunction (Cheong et. al., 2016). These observations provide evidence that βARs can directly be regulated by hypoxia but less is known about the underlying mechanisms. We postulated that hypoxia shifts the homeostasis between kinase and phosphatase driven mechanisms may underlie βAR dysfunction. β2AR HEK 293 cells were exposed to hypoxia (2% O
2
) and assessed the mechanisms underlying desensitization (G-protein coupled receptor kinases, GRKs) and resensitization (Protein phosphatase 2A, PP2A). Six hours of hypoxia treatment resulted in increase of βAR phosphorylation and GRK2 expression, and interestingly, the internalization of phosphorylated β2AR is β-arrestin independent. Assessment of βAR phosphorylation in the plasma membrane and endosomal fractions surprisingly, showed marked increase in β2AR phosphorylation in the endosomal fraction. Furthermore, we also observed that receptor associated PP2A activity was inhibited in the endosomes following hypoxia with minimal changes of activity at the plasma membranes. At the same time, PI3Kγ activity markedly upregulated in the endosomes along with an increase of I2PP2A phosphorylation. Similarly subjecting normal mice to 20 hours of hypoxia resulted in significant cardiac dysfunction (% FS: Normoxia 38.83% vs. Hypoxia 32.38%, P=0.0055; % EF: Normoxia 69.71% vs. Hypoxia 60.76%,P=0.0105) and was associated with significant increase in β2AR phosphorylation associated with significant loss in βAR function as measured by G-protein coupling adenylyl cyclase activity. Given that β-blockers confer beneficial effects, we tested whether β-blocker (propranolol) would prevent βARs phosphorylation under hypoxia or normoxia. Consistently, β-blocker treatment in normoxia results in increased β2AR phosphorylation however, remarkably β-blocker treatment in hypoxia results in loss of β2AR phosphorylation, reduction in GRK2 expression and increase in βAR-associated PP2A. These studies show that agonist-independent hypoxia-driven β2AR dysfunction can be ameliorated by β-blockers and the underlying mechanisms for
this unexpected findings
will be discussed in the presentation. These findings have significant clinical implications as understanding these mechanisms could provide novel insights into the benefits provided by β-blockers.
Abstract 409: Investigating the Role of the Mitochondrial Calcium Uniporter (mcu) in Calcium Regulation of Cardiac Mitochondria Using Transmural Spectroscopy and an Integrating Sphere
Authors: Kosmach Anna , Sun Junhui , Femnou Armel , Balaban Robert S , Murphy Elizabeth
Published: October 2020

Cardiac mitochondria uptake calcium through the mitochondrial calcium uniporter (MCU). To better understand the role of MCU and mitochondrial calcium in regulating heart physiology and pathophysiology, we developed a method to measure mitochondrial matrix calcium in beating, perfused hearts. Langendorff perfusing hearts are loaded with 4.5 uM rhod-2-AM at 30°C and then perfused at 37°C to washout uncleaved dye. We determined that rhod-2 localized primarily to the mitochondria under our loading conditions, as shown by loading in a heart expressing a GFP-tagged mitochondrial outer membrane protein and analyzing heart slices under super resolution microscopy. Further, addition of Mn
2+
, which quenches cytosolic rhod-2, has little effect on rhod-2 signal. We insert an optical catheter with both white light and 532nm laser into the left ventricle and interleaved the collection of spectra from both light sources. The perfused heart is center mounted in an integrating sphere for spectra collection. The light passes through the ventricle and reflects off the integrating sphere resulting in a near uniform sampling of the transmitted light. Spectral properties from both light sources are determined using a rapid scanning spectrophotometer. Myoglobin oxygenation, cytochrome redox state, and rhod-2 loading are determined by white light absorbance. Ca
2+
bound rhod-2 emission is determined by removing background tissue effects from laser emission spectra and normalizing to tissue absorbance. Using this method we are able to measure changes in Ca
2+
and cytochromes during treatments such as isoproterenol and ischemia-reperfusion. The use of an integrating sphere transmural spectroscopy provides us an unique method to study mitochondrial Ca
2+
signaling in perfused mouse heart loaded with Rhod-2.