bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P329: Exosomes Derived From Podoplanin Positive Cells Alter The Cellular Composition Of Healthy Mouse Hearts
Authors: Cimini Maria , Garikipati Venkata , Elia Andrea , Wang Chunlin , TRUONGCAO MAY , Lucchese Anna Maria , Huang Grace , Mallaredy Vandana , Gonzalez Carolina , Benedict Cindy , Kishore Raj
Published: November 2021

Fibrosis and blood hypoperfusion stimulated by paracrine signals enhances the ventricular dysfunctionafter myocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN) positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells could independentlyalter healthy heart physiology and structure. PDPN+ cells were isolated 2 days after MI, culture expandedand activated with TNFα and Angiotensin II. Exosomes derived from activated PDPN+cells conditionedmedia (PDPN+exo) were used in vitro for the treatment of mouse cardiac endothelial cells (mCECs) andmouse fibroblast (3T3) and in vivo for the treatment of healthy mouse hearts. In vitro, PDPN+exoinfluenced the phenotype of mCECs, stimulating their lineage into lymphatic endothelial cells andfacilitated fibroblasts transition to myofibroblast. Characterization of the protein content of PDPN+exoshowed high concentration of Notch receptors and γ-Secretase, suggesting these cellular transitions maydepend on exosome-mediated Notch translocation and cleavage. In fact, after exosomes treatmentcleaved notch (NICD) translocated in the nuclei of mCECs and 3T3 as early as 1h of treatment and eitherHes-1 or Hey-1, major transcription factors activated by NICD were enhanced within 2d of treatment.Using DAPT, a γSecretase inhibitor, notch cleavage was inhibited, and no phenotype switching in responseto exosome treatment was observed. In vivo, PDPN+exo were injected into the left ventricle of healthymouse hearts followed by boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase of the enddiastolic and systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Podoplanin positive cellsrepresent 80% of the scar’s cells of a chronic infarcted myocardium and the specific exosomes cargo highlyinfluence the lineage of cardiac cells altering the biology of endothelial cells and fibroblasts which mayfacilitate adverse remodeling.
Abstract P337: CD36 Is Required To Prime Cardiomyocytes For Proliferation
Authors: Salama Aboubakr M , Abouleisa Riham , Ou Qinghui , Gibreil Ahmed , Mohamed Tamer
Published: November 2021

Many approaches have been explored to regenerate the heart muscle followingischemic injury, out of which is the induction of cardiomyocytes proliferation. Ourprevious work demonstrated that cell-cycle was successfully induced incardiomyocytes by viral expression of combination of four cell-cycle factors: cyclinB1, CDK1, cyclin D1 and CDK4; termed as 4 factors (4F). However, only 15-20 % ofthe cells expressing the four factors progress into cell-cycle, while the remainderare quiescent. A pertinent question in the field of cardiac regeneration is why allviral or in vivo transgenic approaches to induce adult cardiomyocyte proliferation,e.g., 4F, YAP, and CyclinA2, promote proliferation in only a subset ofcardiomyocytes. This general finding suggests that factors or conditions beyondcell-cycle induction influence the probability and perpetuation of cardiomyocytedivision.Here we aim to investigate why only a subpopulation of cardiomyocytes is able toprogress through cell-cycle.Temporal single cell RNA sequencing of 60 days mature hiPS-CMs 24, 48 and 72hours post infection with 4F or control virus revealed that a unique population ofcardiomyocytes in the LacZ control group [1026 cells out of 6761 cells (~15%)] thatdisappears after treatment with 4F; another unique cluster with similar cellnumbers appears 24 h after 4F transduction (Cluster 3), which suggests that theinitial population was primed to proliferate. One of the major characteristics of thisprimed subpopulation is the expression of CD36, a major fatty acids transporter incardiomyocytes, and that 4F induction of cell-cycle completely abolishes CD36expression. knocking down CD36 in hiPS-CMs for 48 hours followed by induction ofproliferation using 4F led to 50-70% reduction in proliferation capacity of thecardiomyocytes compared to the control cells. Furthermore, cardiomyocytesisolated at P7 from CD36 knockout mice showed 30-40 % reduction incardiomyocytes proliferation capacity at baseline and after 4F induction ofproliferation compared to cardiomyocytes isolated from WT controls.These findings suggest that CD36 is needed to prime the CMs to proliferate, andthis can be, at least partially, through provision of energy requirements throughβ-oxidation of fatty acids.
Abstract P338: Myopeptide Improves Contractile Mechanics In A Mouse Model Of Heart Failure Expressing Phosphoablated Cardiac Myosin Binding Protein-C
Authors: Singh Rohit , Sadayappan Sakthivel
Published: November 2021

Rationale:
Normal heart function depends on cardiac myosin binding protein-C (cMyBP-C) phosphorylation. Its decrease is associated with heart failure (HF) by inhibiting actomyosin interactions. In absence of cMyBP-C phosphorylation, the protein is bound to myosin S2, but released when phosphorylated, allowing myosin to form cross-bridges with actin. Challenging cMyBP-C/myosin S2 interaction by myopeptide (the first 126 amino acids of myosin S2) could promote actomyosin interaction
in vitro
, but its ability to improve contractility in HF remains untested.

Objective:
To test contractile function in skinned papillary fibers of a cMyBP-C dephosphorylated mouse model using myopeptide.

Methods and Results:
To mimic constitutive phosphoablation, a knock-in mouse model was established to express cMyBP-C in which serines 273, 282 and 302 were mutated to alanine (cMyBP-C
AAA
). Western blotting revealed 50% and 100% of cMyBP-C
AAA
in het and homo mouse hearts, respectively. Echocardiography showed a decreased percentage of ejection fraction (28%, p<0.01) and fractional shortening (30%, p< 0.05) in both het and homo cMyBP-C
AAA
mice at 3 months of age, compared to knock-in negative controls. These mice also developed diastolic dysfunction with elevated ratio of E/A and E/e’ waves. Next, pCa-force measurements using skinned papillary fibers determined that maximal force (F
max
) and rate of cross-bridge formation (
k
tr
) were decreased in the cMyBP-C
AAA
groups, compared to the control. However, administration of dose-dependent myopeptide increased F
max
and
k
tr
in wild-type and cMyBP-C
AAA
permeabilized skinned papillary fibers without affecting myofilament Ca
2+
sensitivity.

Conclusions:
Myopeptide can increase contractile force and rate of cross-bridge formation by releasing cMyBP-C/myosin S2 and promoting actomyosin formation of cross-bridges, thus validating its therapeutic potential.
Abstract P339: Examining Cardiac Phenotypes Of EF Hand Containing Domain 1 Protein (EFHD1) Knockout Mice
Authors: Eberhardt David R , Yin Xue , Balynas Anthony , Lee Sandra , Walsh Maureen , Funai Katsuhiko , Chaudhuri Dipayan
Published: November 2021

Mitochondrial dysfunction due to calcium overload is common in heart disease and results in heart failure. In a mouse model of mitochondrial cardiomyopathies we noted a substantial downregulation of the gene encoding for the EF hand containing domain 1 protein (EFHD1). EFHD1 is a mitochondrial protein which binds calcium and has been shown to stimulate both apoptosis and mitoflashes. These processes involve mitochondrial calcium overload. We therefore hypothesized that EFHD1 down regulation could represent a natural response by failing hearts to prevent mitochondrial calcium overload. We therefore studied the calcium handling properties of mice where Efhd1 had been knocked out (Efhd1-KO). These mice are viable and have no obvious adverse cardiac or metabolic phenotypes. We find that EFHD1 is expressed in the cytoplasm and mitochondrial outer membrane and intermembrane space. It associates strongly with endoplasmic reticulum (ER)-associated mitochondrial membranes. Moreover, mitochondria isolated from Efhd1-KO mice also exhibited 250±10 % improvement in calcium retention in liver but not heart, while neonatal mouse hearts were 3 times more resistant to hypoxia. These findings suggest that EFHD1 may modulate mitochondrial calcium uptake.
Abstract P346: Ferroptosis Is Critical In Diabetic Atherosclerosis Through Heme Oxygenase 1 Regulated Lipid Peroxidation Pathway
Authors: Meng Zhijun , Zhang Zhen , Tsukuda Jumpei , Ma Xin L , Wang Yajing
Published: November 2021

Introduction:
The patients with diabetes dramatically accelerates atherosclerosis. Although improved diabetes treatment strategy has prevented the pathogenesis, diabetic atherosclerosis is still the main cause of morbidity and mortality. It is urgent to clarify its in-depth mechanism and identify new therapeutic targets for preventing diabetic atherosclerosis.

Hypothesis:
We assessed the hypothesis that ferroptosis and HMOX1 may be implicated in diabetes atherosclerosis through comprehensive in-depth bioinformatics analysis.

Methods and Results:
Analysis of Gene expression profiles (GSE30169 and GSE6584) provided a total of 91 differentially expressed genes (DEGs) including 68 up-regulated and 23 down-regulated genes (A). Ferroptosis, a type of programmed cell death dependent on iron, and HMOX1, a gene that encodes heme oxygenase, were identified as key factors in diabetes atherosclerosis (B). To explore the potential mechanism, we measured iron content and reactive oxygen species (ROS) generation and conducted a lipid peroxidation assay. Compared with animals on a normal diet (ND), ferroptosis and HMOX1 expression in ApoE
-/-
mice on high-fat diet (HFD) were significantly increased (C, n = 3). Ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively attenuated the diabetic atherosclerosis area in HFD animals (D, n = 5, P < 0.01 vs HFD). In addition, Fer-1 restored the ferroptosis-related protein (GPX4 and SLC7A11) with high glucose high lipid (HGHL) treatment in Mouse arterial endothelial cells (MAECs) (E, n=6). More importantly, HMOX1 deficiency rebalanced Fe
2+
overload, decreased iron content, reduced ROS, and alleviated the lipid peroxidation, which led to the reduction of ferroptosis in diabetic endothelial cells (F).

Conclusions:
We demonstrate that ferroptosis is critical in diabetic AS progression and HMOX1 is an implicated factor responsible for ferroptosis in diabetic AS development, suggesting HMOX1 may serve as a potential therapeutic or drug development target.
Abstract P354: Circulating Interleukin 17 A In Patients With Acute And Chronic Coronary Syndrome
Authors: Oliveira Dinaldo C , Mendes Filho Edivaldo , Barros Mariana , Oliveira Carolina , Cabral Joao Vitor , Ferreira Esmeralci
Published: November 2021

Introduction:
Interleukin L-17 is produced by Th 17 cells and other cells. There is a debate if IL 17 is atherogenic or atheroprotective. The true role of this interleukin during the development and progression of the coronary artery disease is not known.

Objective:
To evaluate if there are differences between the IL17 A serum levels according to clinical presentation of the coronary artery disease.

Methods:
This is a cross sectional study which enrolled 101 patients with acute coronary syndrome (ACS), 100 patients with chronic coronary syndrome (CCS) and 100 healthy volunteers. Blood samples were taken from patients ( at admission) and controls to analysis the level of IL17A. Clinical characteristics were collected through questionnaires. This research was approved by ethical committee.

Results:
Comparisons of the clinical characteristics between patients with ACS and CCS revealed: mean age
(
62 ± 12.4 vs 63.3 ± 9.8, p = 0.4
),
male (63.4% vs 58%, p = 0.4) hypertension (85.1% vs 79%, p = 0.1)
,
disyipidemia (48% vs 31%, p =0.01), Diabetes Mellitus (47.5% vs 41%, p = 0.3), previous myocardial infarction (57.4% vs 40%, p = 0,01), smoking (29.7% vs 38%, p = 1). The peripheral concentrations of IL17A according to ACS, CCS and controls were: 5.36 ± 8.83 vs 6.69 ± 17.92 vs 6.26 ± 11.13, p = 0.6. Besides, the comparison between ACS and CCS showed: 5.36 ± 8.83 vs 6.69 ± 17.92, p = 0.3.

Conclusion:
The main finding os this study was that the circulating IL 17 concentrations were similar in patients with ACS, CCS and healthy volunteers). Besides, there was no difference between patients with ACS and CCS. Therefore, our hypothesis is that in patients with ACS and CCS the circulating IL 17 A concentrations are low or undetectable.
Abstract P366: The Impact Of Estrogen Signaling On Gut Epithelial Cells
Authors: Solomon Alice E , Hoyer-Kimura Christina , Kangath Archana , Duca Frank , Konhilas John P
Published: November 2021

Cardiovascular disease (CVD) is the leading cause of death in women worldwide. Menopause is associated with increased susceptibility to CVD, but the mechanism is currently unknown. Studies have suggested that loss of estrogen signaling in the intestine during menopause may lead to worse myocardial infarction (MI) outcomes in female mice. Our lab has shown that suppressing inflammation in the gut using pre- and pro-biotics protects the heart from ischemic heart disease caused by MI through unknown means. We propose that alterations in gut microbiota are due to destruction of intestinal pattern recognition receptors (PRRs) such as toll-like receptor 4 (TLR4) and TLR5. Studies show that estrogen signaling modulates PRRs in other tissues, but this has yet to be investigated in gut epithelial cells. To elucidate the mechanisms of estrogen receptor function in the gut as a direct mediator of vascular health, we are utilizing an immortalized human cell line of colorectal adenocarcinoma brush border expressing (Caco2 BBE) cells.
We hypothesized that estrogen treatment in gut epithelial cells will lead to increased expression of PRRs and decreased inflammation in Caco2 BBE cell lines.
To investigate expression and localization of estrogen receptors (ERs), Caco2 BBE cells at different confluency were treated with estradiol (E2) or DMSO control for 24hrs, then either RNA or protein were harvested for use in qPCR and western blot, or cells were formalin fixed for use in immunohistochemistry. In each case, ERα was not detected, but ERβ was present at varying concentrations. Data also showed increased expression of TLR5 and TLR7 in E2 treated cells, while TNF-α and IL-1 were decreased. In conclusion, data show potential confluency dependent ERβ expression and suggest that ERα may not be present in our Caco2 BBE cell line. Our hypothesis is also supported by data for PPRs and inflammatory markers. Though these results are revealing, future studies are needed to further characterize Caco2 BBE cells in the context of ER signaling.
Abstract P368: Caveolin-3 Prevents Swelling-induced Cell Lysis Via Regulation Of
I
Cl,swell
Expression And Activity
Authors: Turner Daniel G , Tyan Leonid , Stroebel Sami , DeGuire Frank , Lang Di , Glukhov Alexey
Published: November 2021

Background:
Caveolae membrane structures harbor mechanosensitive chloride channels (MCCs) which form a swelling-activated chloride current (
I
Cl,swell
) and play an important role in cell volume regulation and mechano-electrical signal transduction. However, the role of muscle-specific caveolar scaffolding protein caveolin-3 (Cav3) in regulation of MCCs expression, activity, and contribution to cell viability in response to mechanical stress remains unclear. We hypothesized that Cav3-based mechano-protection is enabled by complimentary expression of MCCs.

Methods and Results:
Experiments were performed on native (Cav3-) and Cav3-transfected (Cav3+) HEK-293 cells. Cell stretch was mimicked by light (220 mOsM) or extreme hypoosmotic swelling (<20mOsM). Cav3+ HEK-293 cells were significantly resistant to extreme hypotonic solutions (15 minute incubation) compared to Cav3- HEK-293 cells, and this mechano-protection was significantly reduced when exposed to
I
Cl,swell
selective inhibitor DCPIB (1 μM). We found that three MCCs (ClC-2, ClC-3, and SWELL1, also known as LRRC8A) contain caveolin-binding motifs in their structure, indicating their possible localization in caveolae structures. Co-IP analysis confirmed association of SWELL1 with Cav3. Interestingly, Cav3+ HEK-293 cells showed a significant (by 2-fold) increase of SWELL1 protein level, while ClC-2/3 protein levels remained unchanged. This was accompanied by a 2-fold increase of
I
Cl,swell
, but no change in mRNA expression levels. FRET analysis showed a <10 nm membrane and intracellular association between Cav3 and tested MCCs. Furthermore, Cav3/SWELL1 membrane FRET efficiency was halved in light hypoosmotic solution, as well as after disruption of caveolae structures via cholesterol depletion by 1-hour treatment with 10 μM methyl-β-cyclodextrin. Cav3/ClC-2/3 average membrane FRET efficiency remained unchanged in hypotonic solution.

Conclusions:
We concluded that of MCCs tested, SWELL1 abundance and activity is regulated by Cav3 and that their association relies on membrane tension and caveolae integrity. The present study highlights the mechano-protective properties of Cav3 which are partially facilitated by complimentary SWELL1 expression and activity.
Abstract P380: Common And Distinct Signatures Of Interstitial And Perivascular Fibrosis In Mouse Models Of Hypertensive Heart Disease
Authors: Thisted Louise , Correia Claudia , Jennbacken Karin , Wagberg Maria , Wichern Franziska , Madsen Martin , Zhang Chen , Skytte Jacob L , Hansen Henrik H , Zois Nora E , Vrang Niels , Jelsing Jacob , Roostalu Urmas , Wang Qing Dong
Published: November 2021

Fibrosis is the hallmark of hypertensive heart disease and heart failure with preserved ejection fraction. Perivascular fibrosis impairs vascular function while interstitial fibrosis leads to compromised cardiac contractility. How these fibrosis types are represented in mouse models of hypertensive heart disease and to what extent the transcriptional signatures of cardiac fibrosis are defined by their location is unknown. Mice were dosed over 4 weeks with angiotensin II (AngII) alone or together with α
1
-adrenergic agonist phenylephrine (PE) and were characterized by echocardiography, light sheet imaging and fibrosis histology. While both groups developed systolic and diastolic dysfunction, hypertrophy and perivascular fibrosis, co-administration of PE resulted in a more severe disease phenotype and prevalent interstitial fibrosis, highlighting the benefits of this model in preclinical research. High-precision spatial transcriptomics based on laser capture microdissected perivascular and interstitial fibrotic areas revealed activation of distinct pro-fibrotic as well as cardioprotective pathways in the AngII+PE infusion model. Perivascular and interstitial fibrosis showed remarkable differences in global gene expression signatures, as demonstrated by high expression of osteochondrogenic genes and markers of secretory fibroblasts in perivascular fibrosis. A limited number of upregulated genes is shared between the fibrosis locations. These data collectively show the suitability of mouse models of hypertensive heart disease to study cardiac fibrosis and demonstrate how progression of fibrosis in mice is closely coupled to deteriorating cardiac dysfunction associated with highly distinct molecular signatures of perivascular and interstitial fibrosis.
Abstract P390: Gender And Strain Background Affect Cardiopulmonary Function In

Col4a3
-/-

Alport Mice
Authors: Iansen Irion Camila , Williams Monique , Eisenberg Trevor , Seo Grace , Lambert Guerline , Takeuchi Lauro M , Young Karen C , Hare Joshua M , Shehadeh Lina A
Published: November 2021

Background:
Alport syndrome (AS) is a hereditary form of chronic kidney inflammation that results in renal failure (RF). Our group previously reported that
Col4a3
-/-
mice, a model of AS and RF, on 129J background exemplified multiple features of heart failure with preserved ejection fraction (HFpEF). This study investigates the effect of 3 different genetic backgrounds on cardiopulmonary function in
Col4a3
-/-
mice.

Methods:
Male and female Col4a3
-/-
(Alport) and WT mice on 3 different genetic backgrounds, 129x1/SvJ, C57Bl/6 and BALBC, were examined using echocardiography, whole body plethysmography (WBP) and pressure-volume (PV) loop analysis. Ttest was used for statistical analysis. Four groups per strain were used (n= 4 to 15 per group).

Results:
Compared with their respective age-matched WT controls, both male and female 8-week-old

Col4a3
-/-

129x1/SvJ mice demonstrated impaired systolic function (EF of 56% to 43.7%, p=0.0018 for females and 50.9% to 42% for males, p=0.044; SV, CO, p<0.05) and diastolic dysfunction (increased IVRT from 15.32ms to 21.72ms, p=0.0009 for females; 16.57ms to 26.21ms, p<0.0001 for males; increased MPI, P<0.05). Additionally, PV loop analysis showed an increased EDPVR and end diastolic pressure, prolonged time constant of LV relaxation, and decreased CO and SV (p<0.05). However for

Col4a3
-/-

BalbC (8-week-old) and

Col4a3
-/-

C57Bl/6 (20-week-old) strains, only females exhibited systolic (BalbC: EF% 51% to 45.66%, p=0.106 for females and 43.5% to 40.8% for males, p=0.508; B6: EF% 51.30% to 40.80% p=0.024 for females and 48.43% to 40.91%, p=0.3098 for males; SV, CO, p<0.05) and diastolic dysfunction (IVRT from 15.96ms to 24.91ms for Balb/C and 18.20ms to 25.63ms for B6, p<0.05). Male

Col4a3
-/-

BalbC had an increase in minimum pressure and end-systolic pressure, and an increased pressure at dV/dt-max. Male

Col4a3
-/-

C57Bl/6 mice had an increased ESPVR slope (p<0.05). All strains except BalbC males demonstrated alterations (p<0.05) in pulmonary function including decreased ventilation rate (MV, mL/min) and respiratory frequency (BPM), decreased peak expiratory flow, EF50, increased expiratory time, relaxation time, inspiratory time, and time of pause at end-of-expiration.

Conclusion:
Both male and female

Col4a3
-/-

129x1/SvJ mice demonstrated impaired cardiopulmonary viability. However,

Col4a3
-/-

mice on BalbC or C57Bl/6 strains had cardiopulmonary deficits only in females.
Abstract P393: Fabrication Of Immunomodulatory Hydrogels For Cardiac Repair After Acute Myocardial Infaction
Authors: Yan Weiang , Rafieerad Alireza , Srivastava Abhay D , Alagarsamy Keshav Narayan , Arora Rakesh C , Dhingra Sanjiv
Published: November 2021

Introduction:
The balance of pro- and anti-inflammatory processes is tightly linked to left ventricular remodeling after myocardial infarction. Immune activation also plays a key role in rejection of transplanted allogeneic stem cells. In this study, we present the design, fabrication and characterization of immunomodulatory chitosan-based hydrogels for cardiac repair after myocardial infarction.

Methods:
Chitosan hydrogels conjugated with small immunomodulatory molecules were synthesized through a thermogelation process. Resultant hydrogels were characterized using scanning electron microscopy and Fourier-transformed infrared spectroscopy. Human mesenchymal stem cells were encapsulated into the hydrogels and biocompatibility was assessed after one week using fluorescence microscopy and a colorimetric assay. Immunomodulatory activity was assessed after co-culture with human T-lymphocytes using flow cytometry for CD4+IFN-γ+ pro-inflammatory and CD4+CD25+FoxP3+ regulatory T-lymphocytes.

Results:
Small immunomodulatory molecules were successfully integrated into chitosan hydrogels. Physico-chemical characterization revealed no significant changes to the 3D structure and porosity of hydrogels. The addition of 10μM atorvastatin or 10μM rosuvastatin did not result in significant cytotoxicity to encapsulated mesenchymal stem cells at 3 or 7 days. Addition of statins resulted in marked suppression of CD4+ T-lymphocyte proliferation (Control 25.1 Fold, Atorvastatin 1.0 Fold, Rosuvastatin 2.3 Fold, p<0.001) and activation (CD4+IFN-γ+ Population: Control 87.1%, Rosuvastatin 23.7%, p<0.001) after stimulation. No differences were seen in percentages of CD4+CD25+FoxP3+ regulatory T-lymphocytes (Control 5.5%, Rosuvastatin 5.7%, ns).

Conclusion:
A biocompatible immunomodulatory hydrogel was created through integration of atorvastatin and rosuvastatin into a chitosan hydrogel. Experiments are currently underway
in vivo
to examine its usefulness for stem cell delivery and reducing adverse left ventricular remodeling after myocardial infarction.
Abstract P399: Endothelial Myc Deletion Reduces Doxorubicin-induced Cardiac Dysfunction And Remodeling
Authors: Machi Jacqueline F , Morales Alejo A , Qi Yue , Mendez Nikole Q , Schmidt Augusto F , Rodrigues Claudia O
Published: November 2021

Cardiotoxicity is the most common complication in patients undergoing chemotherapy, and the leading cause of death in long-term cancer survivors. Anthracyclines are highly effective drugs broadly used in cancer treatment, but with limited use due to cardiotoxicity. Currently, there is no effective treatment for anthracycline-induced cardiotoxicity. Recent studies have shown the role of endothelial cells in cardioprotection after anthracycline exposure, though mechanisms involved remain elusive. In the present study, we provide supporting evidence that endothelial Myc plays a role in anthracycline-induced cardiotoxicity. Myc is a stress sensor and master regulator involved in multiple cellular functions. Deregulated Myc expression has been linked to multiple pathological conditions. Using a conditional knockout model, we tested the hypothesis that loss of endothelial c-Myc is cardioprotective. Control (CT) and endothelial knockout (KO) juvenile mice were exposed to 15mg/kg cumulative dose of doxorubicin administered in 6 doses every 2-days along 2-weeks. Echocardiography was performed 3-days after last dose. Doxorubicin had a significant sex-specific impact in cardiac function as previously reported. In CT males, doxorubicin caused significant decline in stroke and diastolic volume, without changes in ejection fraction. In CT females, doxorubicin caused significant increase in heart rate, decline in ejection fraction, and increase in diastolic volume. KO animals showed attenuated response to these doxorubicin-induced alterations in both males and females. Pathology analysis of the heart 3-months after doxorubicin exposure showed significant decline in CT and KO male cardiac mass and collagen content, both of which were attenuated in KO animals. Doxorubicin did not show any impact on mass and collagen content in females. RNAseq analysis suggested modulation of distinct pathways in CT and KO hearts associated with inflammation, DNA damage and repair, cell death and fibrosis. Cdkn1a and TP53 were top upstream regulators predicted to be activated in CT and let-7 in KO hearts. Our findings support the potential targeting of endothelial Myc in doxorubicin-induced cardiotoxicity.
Abstract P401: The Anti-arrhythmic Effects Of VDAC Isoforms Are Mediated By Glutamate 73 Through Regulation Of Mitochondrial Calcium Transport
Authors: Crisman Lauren , Shimizu Hirohito , Langenbacher Adam , Huang Jie , Wang Kevin , Schredelseker Johann , Chen Jau-Nian
Published: November 2021

Mitochondria critically regulate cellular processes such as bioenergetics, metabolism, calcium homeostasis and apoptosis. VDAC proteins are abundant proteins that control the passage of ions and metabolites across the outer mitochondrial membrane. We have previously shown that activation of VDAC2, is able to buffer excess calcium and thereby suppress calcium overload induced arrhythmogenic events in vitro and in vivo. However, the mechanism by which VDAC2 regulates calcium transport and cardiac contractions remained unclear. It is also unclear whether all three VDAC isoforms (VDAC1,2 and 3) possess similar cardioprotective activity. The zebrafish
tremblor/ncx1
mutant lacks functional NCX1 in cardiomyocytes leading to calcium overload, and the manifestation of fibrillation-like phenotypes. Using the
tremblor/ncx1
mutant as a model, we observed isoform-specific differences between the VDAC family members. VDAC1 and VDAC2 enhanced mitochondrial calcium trafficking and restore rhythmic contraction in
tremblor
mutants, whereas, VDAC3 did not. We found that the differing rescue capabilities of VDAC proteins were dependent upon residues in their N-terminal halves. Phylogenetic analysis further revealed the presence of an evolutionarily conserved glutamate at position 73 (E73) within VDAC1 and VDAC2, but a glutamine (Q73) in VDAC3. Excitingly, we showed that replacing VDAC2 E73 with Q73 ablated its calcium transporting activity. Conversely, substituting the Q73 with E73 allows VDAC3 to gain calcium trafficking and cardioprotective abilities. Overall, our study demonstrates an essential role for the evolutionarily conserved glutamate-73 in determining the anti-arrhythmic effect of VDAC isoforms through their regulation of mitochondrial calcium uptake.
Abstract P405: Cardioprotective Actions Of Nitroxyl Donor Angeli's Salt Are Preserved In The Diabetic Heart And Vasculature In The Face Of Nitric Oxide Resistance
Authors: Velagic Anida , Li Jasmin Chendi , Qin Chengxue Helena , Li Mandy , Deo Minh , Marshall Sarah A , Woodman Owen L , Horowitz John D , Kemp-Harper Barbara K , Ritchie Rebecca H
Published: November 2021

Introduction:
The risk of fatal cardiovascular events is increased in patients with type 2 diabetes mellitus (T2DM). A major contributor to poor prognosis is impaired nitric oxide (NO•) signalling at the level of tissue responsiveness, termed NO• resistance. Nitroxyl (HNO) induces positive inotropic and lusitropic effects in healthy and failing hearts.

Hypothesis:
We hypothesised that in a rodent model, T2DM will promote, and HNO will circumvent, NO• resistance in the myocardium and coronary vasculature.

Methods:
At 8 weeks of age, male Sprague-Dawley rats commenced a high-fat diet. After two weeks, the rats received low-dose streptozotocin (two intraperitoneal injections, 35 mg/kg, over two consecutive days), and continued the diet. Twelve weeks later, hearts were Langendorff-perfused to assess responses to the NO• donor diethylamine NONOate (DEA/NO) and the HNO donor Angeli’s salt.

Results:
Inotropic, lusitropic and coronary vasodilator responses to DEA/NO were impaired, and responses to Angeli’s salt were preserved or enhanced, in T2DM hearts compared with non-diabetic hearts.

Conclusions:
This is the first evidence that inotropic and lusitropic responses are preserved, and NO• resistance in the coronary vasculature is circumvented, by the HNO donor Angeli’s salt in T2DM. These findings highlight the cardiovascular therapeutic potential of HNO donors, especially in cardiac emergencies such as acute ischaemia or heart failure.

Figure 1.
Dose-response curves and maximal responses to DEA/NO or Angeli's salt in diabetic or non-diabetic hearts. (A-C) LV+dP/dt, (D-F) LV-dP/dt and (G-I) coronary flow rate. Data expressed as change from baseline (denoted by Δ), mean ± SEM. Data analysed by two-way RM ANOVA with Sidak's post-hoc test. *P<0.05 vs. non-diabetic. LV, left ventricular; LV+dP/dt, maximal rate of rise in LV pressure; LV-dP/dt, maximal rate of fall in LV pressure.
Abstract P410: A Novel Curcumin Formulation, ASD-cur Suppressed Heart Failure After Myocardial Infarction In Rats
Authors: Takai Hidemichi , Morimoto Tatsuya
Published: November 2021

Introduction:
Curcumin prevents the development of heart failure and is a potential treatment for heart failure. Although curcumin is known to be safe, its therapeutic efficiency is limited due to its low bioavailability. To overcome this problem, we developed ASD-Cur, an amorphous formulation of curcumin. In this study, we investigated the effect of ASD-Cur and compared it with Theracurmin
®
, a colloidal submicron dispersion of curcumin.

Methods:
Male SD rats were orally administrated with ASD-Cur or Theracurmin
®
(10 mg/kg curcumin). The plasma levels of curcumin were measured at 0.25, 0.5, 1, 2, 4 and 6 hours after administration. Twelve healthy volunteers, who had provided written informed consent, were administrated with ASD-Cur and Theracurmin
®
containing 30 mg curcumin, and plasma curcumin concentrations were determined at 0.5, 1, 2, 4, and 8 hours. Next, male SD rats were subjected to MI or sham surgery. One week after surgery, the MI rats were randomly assigned to 4 groups: vehicle, ASD-Cur (0.2 mg/kg curcumin) or Theracurmin
®
(0.2 or 0.5 mg/kg curcumin). Oral administration of these compounds was repeated for 6 weeks. After echocardiographic examinations, myocardial cell diameter, perivascular fibrosis, mRNA levels, and the acetylation of histone H3K9 were measured.

Results:
After administration in rats, the area under the plasma concentration-time curve (
AUC
0-6h
) and the maximum plasma concentration (
C
max
) of ASD-Cur were 3.7-fold and 9.6-fold higher than those of Theracurmin
®
, respectively. The
AUC
0-8h
and
C
max
of ASD-Cur in humans were 3.4-fold and 5.4-fold higher than those of Theracurmin
®
, respectively. Echocardiographic analysis showed that 0.2 mg/kg ASD-Cur and 0.5 mg/kg Theracurmin
®
significantly improved the MI-induced deterioration of FS and left ventricular hypertrophy to the same extent. Both treatments significantly suppressed MI-induced increases in myocardial cell diameter, perivascular fibrosis, mRNA levels of hypertrophic markers and cardiac fibrosis, and acetylation of histone H3K9 to the same extent.

Conclusion:
These findings indicated that ASD-Cur has greater bioavailability than Theracurmin
®
, and could exhibit greater therapeutic potency towards for MI-induced heart failure at a lower dose.
Abstract P411: Sex-based Differences In A Feline Model Of Hfpef
Authors: Eaton Deborah M , Berretta Remus , Lynch Jacqueline E , Travers Joshua G , Woulfe Kathleen C , Hobby Alexander R , Schena Giana , Johnson Jaslyn , Wallner Markus , McKinsey Timothy A , Wolfson Marla R , Houser Steven R
Published: November 2021

Rationale:
Heart Failure with preserved Ejection Fraction (HFpEF) accounts for approximately 50% of all HF diagnoses with no FDA approved therapies. HFpEF is more prevalent in females versus males, but the mechanisms driving the development of HFpEF as a sex-based disorder are not well understood. We have recently shown that slow progressive pressure overload (PO) in male felines induces a HFpEF phenotype but have not investigated the differences in response to the same physiological stress in females.

Hypothesis:
Females will develop a phenotype that is distinct from males in response to PO.

Methods and Results:
Male (m) and female (f) domestic short felines (age 2mo) underwent either a sham procedure (m: n=7; f: n=7) or aortic constriction (m: n=11; f: n=10) using a customized pre-shaped band. At baseline (prior to surgery), there was no difference in body weight between groups and echocardiography revealed no significant difference in the ratio of left atrium to aortic root (LA/Ao), LA ejection fraction (LA EF), left ventricle (LV) ejection fraction, LV wall-thickness, and E/A ratio. At 4mo post-surgery, both males and females developed cardiac dysfunction. Females gained significantly less weight than males throughout the study. Despite the size difference, both sexes developed comparable LV wall thickness and changes in E/A ratio vs. sham groups. There was no change in LV EF. Furthermore, there was a decrease in LA EF and increased LA/Ao, indicating LA dysfunction and enlargement. Invasive hemodynamics at 4mo post-surgery showed no differences between sexes for the systolic pressure gradient generated by the aortic banding. Banded males had a significantly higher LV end-diastolic pressure vs. banded females, but there was a trend towards prolongation of tau and lower dp/dt
min
in banded females, reflective of worse active relaxation. Both sexes had comparable dP/dt
max
. There were no differences between banded males and females in heart weight to body weight or cardiomyocyte cross-sectional area.

Conclusion:
Despite similar pressure gradients as a result of PO and the development of similar cardiac hypertrophy between sexes and a higher LVEDP in males, females had a trend towards worse relaxation. Other causes of HFpEF may have sex-based differences.
Abstract P418: Transcriptomic And Genomic DNA Accessibility Dynamics During Differentiation Of Induced Pluripotent Stem Cells Derived Smooth Muscle Cells And Acquisition Of Contractile Phenotype
Authors: LIU LU , Georges Adrien , Bouatia-Naji Nabila
Published: November 2021

Introduction:
Smooth muscle cells (SMCs) capacity to phenotype switching between proliferative and quiescent (contractile) is a widely studied mechanism in cardiovascular disease. Primary SMCs tend to lose many physiological features in culture, which makes the study of their contractile function challenging. Recently, an optimized protocol of induced pluripotent stem cells (iPSCs) differentiation into contractile SMCs was described. Here we aimed at defining the transcriptomic and open chromatin dynamics during the acquisition of SMCs phenotypes.

Methods:
We differentiated 4 human iPSC lines (2 males, 2 females) towards either contractile (Repsox induced) or synthetic (PDGF-BB/TGF-β induced) SMC phenotypes using a 24-days protocol. We performed RNA-Seq and assay for transposase accessible chromatin (ATAC)-Seq at 5 time points of differentiation. We analyzed gene expression profiles and compared them to existing dataset of human aorta by principle component analyses (PCA) and gene set enrichment analyses using GO terms.

Results:
iPSCs derived SMCs showed expected morphology and positive expression of SMC markers. Synthetic SMCs (SSMCs) exhibited greater capacity of proliferation, migration and lower calcium release capacity, compared to contractile SMCs (CSMCs). RNA-Seq results showed that multiple genes involved in the contractile function of arteries, including myosin light chain kinase
(MYLK)
and angiotensin type 1 receptor (
AGTR1
) genes were highly expressed in CSMCs compared to SSMCs. Overall, CSMCs conserved SMC properties beyond 24 days and their gene expression profile clustered near human aorta. During late differentiation stages, CSMCs showed an upregulation of genes involved in cardiovascular system development, whereas genes involved in cell stress were upregulated in SSMCs.

Conclusions:
We describe global genomic profiles of iPSCs derived CSMCs that presented comparable gene expression profiles to mature artery tissue. Combination with upcoming DNA accessibility maps is expected to allow the functional exploration of genetic risk variation involved in several arterial diseases involving the impairment of the SMCs contractile function.
Abstract P427: Inhibition Of PP2A By Insulin Impairs Beta-Adrenergic Receptor Function
Authors: Sahu Anita , Mukherjee Sromona D , Witherow Conner P , Stenson Kate , Tesmer John , Mohan Maradumane L , Naga Prasad Sathyamangla V
Published: November 2021

Insulin impairs β2-adrenergic receptor (β2AR) function via trans-phosphorylation through G protein-coupled receptor kinase 2 (GRK2). However, less is known about dephosphorylation mechanisms mediated by protein phosphatase 2A (PP2A) during this insulin-β2AR cross-talk. Pharmacologic or genetic inhibition of phosphoinositide 3-kinase γ (PI3Kγ) unexpectedly resulted in significant reduction of insulin-mediated β2AR phosphorylation. Interestingly, β2AR-associated phosphatase activity was inhibited by insulin but was reversed by knock-down of PI3Kγ showing negative regulation of PP2A by PI3Kγ. Co-immunoprecipitation and surface plasmon resonance studies using purified proteins showed that GRK2 and PI3Kγ form a complex and could be recruited to β2ARs as GRK2 interacts with insulin receptor substrate (IRS) following insulin treatment. Further, co-immunoprecipitation studies showed that PI3Kγ directly interacted with both IRS-1 and IRS-2 but only IRS-2 interaction with PI3Kγ significantly increased following insulin stimulation. These results indicated that PI3Kγ could also be directly recruited to the receptor complex by IRS-2. Consistently, β-blocker pretreatment did not reduce insulin-mediated β2AR phosphorylation indicating agonist- and Gβγ-independent non-canonical regulation of receptor function. Mechanistically, PI3Kγ inhibits PP2A activity at the βAR complex by phosphorylating an intracellular inhibitor of PP2A (I2PP2A). Knock-down or CRISPR ablation of endogenous I2PP2A unlocked PP2A inhibition mediating β2AR dephosphorylation showing an unappreciated acute regulation of PP2A in mediating insulin-β2AR cross-talk.
Abstract P446: Cyclophilin D Inhibition Rescues Cardiac Function And Electron Transport Chain Assembly And Activity In Hypoxia Exposed Neonatal Mice
Authors: Burris Jonathan , Beutner Gisela , Porter George A
Published: November 2021

Introduction:
Mitochondria play a critical role in cardiac myocyte physiology and differentiation. Hypoxia decreases cardiac function. Changes in embryonic heart metabolism at the level of the electron transport chain (ETC) are regulated by a chaperone protein known as cyclophilin D (CypD). Inhibition of CypD with chemicals such as cyclosporin A (CsA) and N-methyl-4-isoleucine cyclosporin (NIM811) leads to more complex mitochondrial structure and effective oxidative phosphorylation.

Hypothesis:
Inhibition of CypD with CsA or NIM811 will rescue the detrimental effects of hypoxia on cardiac function and on ETC assembly and activity.

Methods:
Mice were exposed to continuous hypoxia (12% oxygen) immediately before birth (gestational age E19.5) to postnatal day 7 (P7). Hypoxic mice received no treatment (No Tx) or intraperitoneal injections 10mg/kg of vehicle (VEH), CsA, or NIM811 from P1 to P6. Litters of mice born into room air served as controls. On P7, mice were anesthetized and underwent echocardiography and/or hearts were harvested for mitochondrial isolation. Enzymatic activity of ETC complexes was quantified using spectrophotometry and normalized to total protein. To measure physical assembly of complex I & V of the ETC, high resolution clear native polyacrylamide gel electrophoresis (HCRN PAGE) followed by in-gel assays were utilized using appropriate protein loading controls.

Results:
Cardiac ejection fraction was decreased in hypoxic No Tx (P<0.0001) and VEH (P<0.0001), but was rescued by CsA or NIM811 when compared to room air controls (P>0.05). Heart weight to body weight ratio was increased in No Tx and VEH groups (P< 0.0001) and rescued in the CsA and NIM811 groups when compared to room air controls (P>0.05). Complex I enzymatic activity was rescued with treatment with CsA and NIM811. HCRN PAGE followed by in-gel ETC complex assay demonstrated assembly of complexes I and V into dimers and tetramer in the room air, CsA, and NIM811 groups that was not seen in the No Tx and VEH groups.

Conclusion:
Pharmacologic inhibition of CypD reversed the effects of hypoxia on cardiac function and ETC activity and assembly in the neonatal heart. Our studies may help develop therapies to treat neonatal cardiomyopathies and the effects of hypoxia on the neonatal heart.
Abstract P448: Role For Btg1 And Btg2 In Postnatal Cardiomyocyte Cell Cycle Arrest And Maturation
Authors: Velayutham Nivedhitha , van Leeuwen Frank N , Scheijen Blanca , Yutzey Katherine E
Published: November 2021

Background:
Adult mammalian cardiomyocytes (CM) are predominantly post-mitotic and cannot proliferatively repair the heart following myocardial infarction (MI). Overexpression of the transcription factor Tbx20 in adult mouse CMs promotes proliferative cardiac repair post-MI, via mechanisms including direct repression of anti-proliferative genes
p21
,
Meis1
, and
Btg2
. Btg2 (B-cell translocation gene 2), a tumor suppressor and transcriptional co-regulator, exhibits high structural and functional similarity with Btg1. However, both Btg1 and Btg2 (Btg1/2) are virtually uncharacterized in the heart. Here, we investigate the role of Btg1/2 in postnatal cardiac maturation.

Methods and Results:
By immunostaining in embryonic, neonatal, and adult C57BL/6 mouse hearts, the highest expression of Btg1/2 was observed in late fetal and early neonatal ventricles, concurrent with upregulation of other CM cell cycle inhibitors. In neonatal mouse CMs
in vitro,
siRNA-mediated loss of Btg2 leads to increased CM proliferation.
In vivo
, Btg1/2 constitutive single- and double- knockout (SKO and DKO respectively) mice exhibit normal heart weight-to-body weight ratios compared to age-matched wildtype (WT) controls, at postnatal day (P)7, P30, and 1 year after birth. Interestingly, at P7, DKO mice have significantly higher CM mitotic activity, as indicated by pHH3 staining, compared to WT. In addition, DKO mice also exhibit significantly smaller CM cross-sectional area at P7 compared to WT. However, by P15, CM mitotic activity and cell size are comparable between WT and Btg1/2 KO mice. Currently, siRNA-mediated knockdown of Btg1/2 in neonatal rat ventricular cardiomyocyte cultures and RNAseq studies are being performed, to assess the transcriptional regulatory roles of Btg1/2 in rodent CMs.

Conclusions:
Here, we highlight two novel regulators of postnatal CM maturation, Btg1 and Btg2, which are upregulated coincident with CM mitotic arrest in mice. Similar to p21 and Meis1, Btg1/2 depletion in mice induces a brief period of increased CM proliferative activity before onset of CM cell cycle arrest. Our results provide evidence for Btg1/2 working in tandem with other cardiac transcription factors and cell cycle regulators, to control CM mitotic arrest after birth.
Abstract P458: Myosin Light Chain Dephosphorylation By Ppp1r12c Promotes Atrial Hypocontractility In Atrial Fibrillation
Authors: Gonzalez-Gonzalez Francisco J , Perike Srikanth , Damen Frederick , Capote Andrielle , Alsina Katherina M , Levin Benjamin , Sridhar Arvind , Grand Jacob , Martin Jody , Akar Joseph , Warren Chad M , Solaro John , Darbar Dawood , Goergen Craig , Wolska Beata M , Wehrens Xander H , McCauley Mark
Published: November 2021

Introduction:
Atrial fibrillation (AF), is the most common sustained arrhythmia, with an estimated prevalence in the U.S. of 2.7 million to 6.1 million and is predictive to increase to 12.1 million in 2030. AF increases the chances of a thromboembolic stroke in five-fold. Although atrial hypocontractility contributes to stroke risk in AF, the molecular mechanisms reducing myofilament contractile function in AF remains unknown.

Objective:
The overexpression of PPP1R12C, causes hypophosphorylation of atrial myosin light chain 2 (MLC2a), decreasing atrial contractility.

Methods and Results:
Left and right atrial appendage tissues were isolated from AF patients versus sinus rhythm (SR). To evaluated the role of PP1c-PPP1R12C interaction in MLC2a de-phosphorylation we used Western blots, coimmunoprecipitation, and phosphorylation assays. In patients with AF, PPP1R12C expression was increased 3.5-fold versus SR controls with an 88% reduction in MLC2a phosphorylation. PPP1R12C-PP1c binding and PPP1R12C-MLC2a binding were significantly increased in AF. In vitro studies of either pharmacologic (BDP5290) or genetic (T560A) PPP1R12C activation demonstrated increased PPP1R12C binding with both PP1c and MLC2a, and dephosphorylation of MLC2a. Additionally, to evaluate the role of PPP1R12C expression in cardiac function, mice with lentiviral cardiac-specific overexpression of PPP1R12C (Lenti-12C) were evaluated for atrial contractility using echocardiography, versus wild-type and Lenti-controls. Lenti-12C mice demonstrated a 150% increase in left atrium size versus controls, with reduced atrial strain and atrial ejection fraction. Also, programmed electrical stimulation was performed to evaluate AF inducibility in vivo. Pacing-induced AF in Lenti-12C mice was significantly higher than controls.

Conclusion:
The Overexpression of PPP1R12C increases PP1c targeting to MLC2a and provokes dephosphorylation, that cause a reduction in atrial contractility and increases AF inducibility. All these discoveries advocate that PP1 regulation of sarcomere function at MLC2a is a main regulator of atrial contractility in AF.