bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1160: Genome-wide CRISPR Screen Reveals Genetic And Small Molecule Modifiers Of Ca
2+
-mediated Cell Death
Authors: Reyes Gaido Oscar , Granger Jonathan , Schole Kate , Luczak Elizabeth D
Published: October 2023

Ca
2+
is a ubiquitous and highly regulated signaling ion in living cells. Thus, Ca
2+
is typically maintained at a low intracellular concentration (10-100 nM), tens of thousands of times lower than the outside space (1-2 mM). Failure to maintain this homeostasis—due to membrane instability or energetic failure—allows supraphysiologic concentrations to enter cells and trigger cell death pathways including mitochondrial permeability transition, apoptosis, and autophagy. This mechanism of cell death is shared by many conditions including myocardial infarction, stroke, and sepsis. Targeting of known protein effectors downstream of Ca
2+
overload, such as caspases and calpains, has not yet translated into therapies due to specificity concerns. Therefore, there is still a pressing need for a comprehensive examination of genetic effectors involved in Ca
2+
-induced cell death. Here, we performed genome-wide CRISPR knockout screening in human cells and identified genes and pathways previously unknown to mediate cell toxicity after Ca
2+
overload. We reveal 115 gene knockouts that protected cells and 9 knockouts that sensitized cells to intracellular Ca
2+
. Notably, genetic ablation of members from the store operated calcium entry, very long-chain fatty acid synthesis, and SWItch/Sucrose Non-Fermentable (SWI/SNF) pathways provided marked protection. Reassuringly, genes known to maintain Ca
2+
homeostasis (e.g., STIM1, ATP2B4, ATP2B1) were recovered by the screen. By comparing against the essential and druggable genomes, we determined that 82 of the protective knockouts are non-essential genes and 21 are targetable with small molecules. Therefore, a significant portion of our hits represent viable but unexplored targets for conditions that feature Ca
2+
overload. We tested this hypothesis using small molecule blockade of hits overlapping with the druggable genome. We found several antagonists that resulted in a reduction of ionomycin-induced cell death. Altogether, our results reveal numerous novel effectors of Ca
2+
-mediated cell death that will serve as a basis for improved mechanistic understanding of cell death and as targets for pharmacotherapeutic design.
Abstract P1181: Targeted Loss Of Sarcolemmal Membrane Associated Protein Isoform 3 (slmap3) In Cardiac Progenitors Stunts The Embryonic Growth Of Myocardium
Authors: Rehmani Taha , Dias Ana , Salih Maysoon , Tuana Balwant
Published: October 2023

Sarcolemmal membrane associated proteins (SLMAPs) belong to the superfamily of tail anchored membrane proteins known to regulate variety of functions including vesicle transport. SLMAP3 is the largest isoform which is ubiquitously expressed and has been linked to Brugada syndrome and sodium channel activity. SLMAP3 is expressed during cardiogenesis, and we examined its role by nullifying its expression in mouse cardiac progenitors using the Nkx2.5-cre-lox system. Hearts from SLMAP3-knockout (KO) mouse embryos at different stages of cardiogenesis, e9.5, e12.5 and e16.5, were smaller (p<0.05) than wildtype (Wt) littermates. Histological analysis of heart wall measurements from the left ventricles revealed thinner walls in SLMAP3-KO from e9.5 (-25.16%, p<0.05), e12.5 (-29.36%, p<0.05) and e16.5 (-40.27%, p<0.05) compared to Wt although normal chamber formations was observed at these developmental stages. To evaluate the potential reasons for the early difference in cardiac growth, we examined myocardial cell numbers and size with markers of proliferation and hypertrophy. Immunofluorescent analysis of e12.5 hearts with proliferative markers (pH3, Ki67) was not significantly (-5%, p>0.05) altered in KO hearts while cardiomyocyte size differences assessed by immunofluorescence with anti-Troponin C, revealed that KO cardiomyocytes were significantly (-19.4%, p<0.05) smaller than Wt. However, the phospho-to total ratios of AKT1(1.45%, p>0.05) and MTOR1(-12%, p>0.05) were not significantly altered in KO hearts when compared to Wt. SLMAP3 has been implicated in Hippo signaling via the STRIPAK (striatin interacting phosphatase and kinase) complex to impact YAP/TAZ and cell growth. Western blot analysis of e12.5 Wt and KO hearts showed no change in phosphorylation of MST1/2(-6.64%, p>0.05) or YAP-Ser 127(-8.84%, p>0.05) ruling out involvement of Hippo signaling. Interestingly, the expression of the other SLMAP isoforms, SLMAP1 and SLMAP2 remained unaffected during cardiac development in SLMAP3 KO hearts. Thus, SLMAP expression is critical for normal development since the loss of SLMAP3 isoform leads to stunted growth of the embryonic heart. Supported by CIHR.
Abstract P2009: Mice Lacking S-nitrosoglutathione Reductase (gsnor
-/-
), A Mouse Model Of Preeclampsia, Exhibit Abnormal Calcium Regulation And Cardiac Hypertrophy During Pregnancy.
Authors: Dulce Raul A , Balkan Wayne , Hare Joshua M , Kulandavelu Shathiyah
Published: October 2023

Introduction:
We recently showed that mice lacking S-nitrosoglutathione reductase (GSNOR
-/-
mice), a denitrosylase that regulates protein S-nitrosylation, exhibit the clinical features of preeclampsia (PE) a leading cause of maternal mortality. PE predisposes the mother to increased risk of cardiovascular (CV) injury later in life and is linked to cardiac hypertrophy and impaired relaxation. Cardiac hypertrophy and relaxation are regulated by calcium cycling. We hypothesized that GSNOR deficiency in pregnant mice alters calcium cycling, leading to hypertrophy and impaired cardiac relaxation.

Methods:
Pregnant control (WT [C57B6]) and GSNOR

-/-

mice were examined at non-pregnant (NP) and late pregnancy (17.5 dpc). Cardiomyocytes (CMs) (N=3-4 mice/group; n=8-24 CMs/heart) were isolated and assessed for intracellular calcium and sarcomere shortening using IonOptix system. Dynamic parameters associated with calcium decay and sarcomere relaxation were recorded (time to peak (ttp), time to 50% of baseline (tt50%);
d
SL/
d
t
min
, time to
d
[Ca]/
d
t
min
, etc.). Experiments were conducted at 37°C.

Results:
At late pregnancy, systolic calcium levels were significantly elevated in CMs isolated from GSNOR

-/-

as compared to WT mothers. Despite the increased cytosolic calcium, CMs from pregnant GSNOR
-/-
mice exhibited no change in contractility. The improved calcium decay rate observed in CMs from pregnant versus NP WT mice, was absent in GSNOR
-/-
mothers. Similarly, sarcomere relaxation parameters and sarcomere resting length, which were also improved by pregnancy in WT CMs, were impaired in CMs from pregnant versus NP GSNOR
-/-
mice. In addition, hearts were significantly enlarged in GSNOR
-/-
mothers as compared to WT, even when normalized to tibia length.

Conclusion:
The elevated systolic calcium levels, impaired cardiac relaxation, and cardiac hypertrophy seen in pregnant GSNOR

-/-

mice may predisposes them to higher risk for CV injury during pregnancy and later in life.
Abstract P2014: Knockout Of Prostaglandin E2 Ep3 Receptor Improves Cardiac Function In Both Sexes In A Mouse Model Of Myocardial Infarction
Authors: Bryson Timothy D , Bhat Shaheen Y , Taube David , Xu Jiang , Peterson Edward , Harding Pamela
Published: October 2023

Prostaglandin E
2
is an autacoid that acts through 4 G-protein-coupled receptors (EP1-EP4). We previously reported that expression of the EP3

receptor increases after myocardial infarction (MI) and mediates reduced cardiac function. This effect was exacerbated in mice that overexpress EP3 in the cardiomyocytes. Furthermore, we reported that the EP3 receptor antagonist, L798106 reduced blood pressure in the Angiotensin II hypertension model and improved cardiac function. We therefore hypothesized that cardiomyocyte specific knock-out of EP3 (EP3 KO) and/or pharmacological antagonism of EP3 protects the heart from cardiac dysfunction after MI. To test our hypothesis, we created a cardiomyocyte specific (αMHC promoter), tamoxifen inducible CM-EP3 KO mouse. Fifteen-week-old male and female EP3 KO and floxed controls underwent sham or MI surgery. After 2 wks, echocardiography was performed, and all statistical analysis was performed by a biostatistician at Henry Ford Health. There were no significant differences in cardiac function between strains after sham operation. After 2 wks MI, male floxed control mice showed significant reductions in ejection fraction (EF;68 ± 5.6 % sham vs 49.1 ± 5.2 % MI; p<0.05), coupled with increased left ventricle dimension at systole (LVDs;1.12 ± 0.08 mm sham vs 2.08 ± 0.34 mm MI; p<0.05). Remarkably, however, these changes were not observed for EP3 KO mice post-MI. EF was 73.1 ± 0.68% in sham vs 69.0 ± 0.81% for MI; and LVDs was 1.09 ± 0.05 in sham vs 1.09 ± 0.04 for MI. Similarly, EF was 66.3 ± 2.4% in female sham mice and remained unchanged after MI (EF; 62.7 ± 2.3% in EP3 KO MI). To investigate a more translational and therapeutic approach, we examined whether daily administration of L798106 improves cardiac function post MI. Male C57BL/6 mice were subject to MI and assigned to vehicle or L798106 treatment (40 μg/Kg/day, s.c.) beginning 3 days post-surgery. After 2 wks, EF was substantially improved from 36.0 ± 2.6% in vehicle vs 49.4 ± 3.0% in L798106, p<0.01. Similar improvements were observed in fractional shortening. Altogether, these data suggest that EP3 may play a direct role in regulating cardiac function and future experiments are warranted to investigate the use of an EP3 antagonist as a potential therapeutic in heart failure.
Abstract P2018: In Vivo Imaging Of The Coronary Microcirculation Throughout The Contractile Cycle With Optical Coherence Tomography Angiography
Authors: Zhou Yirong , Kim Junyoung , Aguirre Aaron D
Published: October 2023

Introduction:
Studying coronary microvascular disease (CMD) warrants
in vivo
high-resolution microcirculation imaging, which requires advanced motion correction and gating strategies. We hypothesized that cardiac and respiratory gating and custom angiogram reconstruction algorithms may enable label-free optical coherence tomography angiography (OCTA) of the coronary microcirculation
in vivo
in mice throughout the cardiac cycle.

Methods:
C57BL/6 male mice (n=20) were imaged through a sternotomy while anesthetized and ventilated. OCTA was performed using a custom tissue stabilizer and a Thorlabs TEL 321 OCT system, which was synchronized to physiologic signals using custom hardware and software and cardiac pacing protocols (Fig. 1A). Angiograms were created by calculating speckle variance between images with one of two strategies: 1. Images within the same cardiac cycle (AB method); or 2. Images in two different cardiac cycles (AC method). Angiograms were formed during both diastole and systole (Fig. 1C and E). Data were processed in MATLAB and ImageJ.

Results:
Without gating or stabilization, angiograms cannot be produced (Fig. 1B). Using stabilization with custom gating and reconstruction strategies significantly improved image registration (Fig. 1G) and allowed creation of widefield capillary angiograms without exogenous contrast agent during both diastole and systole (Fig. 1D and F).

Conclusion:
In vivo OCTA can image the coronary microcirculation in the heart throughout the cardiac cycle and will provide a powerful new tool for investigating coronary microvascular dysfunction in mouse models of heart disease.
Abstract P2036: Induced Pluripotent Stem Cell- Derived Cardiomyocytes Provide New Insights Of The Pathogenic Consequences Of The Novel Frameshift Variant
(c.5769delG)
In
MYH7
Gene
Authors: Elshorbagy Sara , Ibrahim Ayman M , Mohamed Fiesal , Fourre Jerome , Downing Barrett , Osman Manar , A. Elfawy Hasnaa , Fathy Mariam , Allouba Mona , Aguib Yasmine , Terracciano Cesare M , Yacoub Magdi
Published: October 2023

Background:
the genetic basis of hypertrophic cardiomyopathy is pleiotropic; A novel disease causing frameshift variant (c.5769delG,
MYH7
) in the beta-myosin heavy chain gene has been recently described. However, the mechanisms involved have not been adequately defined. We have used iPSC-CMs to investigate the underlying mechanisms.

Methods:
We have generated patient-specific iPSCs from clinically and genetically defined HCM patients carrying the pathogenic variant (c.5769delG) (n=3) and compared them to iPSCs from healthy controls (n=3). Optical mapping of calcium transients and cell action potential were performed under different stimulation conditions. iPSC-CMs contractility was recorded via widefield microscopy. Gene and protein expression analyses were performed using RT-PCR, immunoblotting and immunocytochemistry. RNA sequencing data from patients’ myocardial tissues was analyzed to validate gene expression data from iPSC-CMs.

Results:
HCM iPSCs-CMs showed a significant decrease in calcium release duration (i.e., time to peak) with decrease in calcium transient amplitude, compared to controls. This was associated with an accelerated contraction and a decreased contraction amplitude. Nonetheless, action potential analysis showed no significant changes. Transcriptome analysis of iPSC-CMs and patients’ myocardial tissues showed a significant down regulation of SERCA/ATP2A2
, MYH6
and
KCNIP2
.

Conclusion:
iPSC-CMs provided new insights of the pathogenic consequences of the novel variant in MYH7 gene on calcium handling and contractility.

Key words:
Hypertrophic Cardiomyopathy, Disease model,
MYH7
, iPSC-CMs, Calcium Handling, Contractility.
Abstract P2062: Distinct Stiffness-induced Mechanical Memory Induction And Maintenance In Cardiomyocytes And Cardiac Fibroblasts
Authors: BOUHRIRA NESRINE , Vite Alexia , Margulies Kenneth B
Published: October 2023

Recognizing that cells “feel” and respond to their mechanical environment, recent studies demonstrate that many cells exhibit a phenomenon of “mechanical memory” in which features induced by prior mechanical cues persist after the mechanical stimulus has ceased. While there is a general recognition that different cell types exhibit different responses to changes in extracellular matrix stiffening, the phenomenon of mechanical memory within myocardial cell types has received little attention to date. To probe the dynamics of mechanical memory in cardiomyocytes(CMs) and cardiac fibroblasts (CFs), we developed a magnetorheological elastomer (MRE) cell culture substrate with tunable and reversible stiffness spanning the range from normal to diseased myocardium. Employing IPSC-derived CMs, and CFs while measuring canonical responses to matrix stiffening: cell area, myofibroblasts activation, and chromatin condensation parameter reveals distinct temporal dynamics of mechanical memory induction in each cell type where mechanical memory is invoked much earlier in CMs than in CFs. Experiments using specific inhibitors of cytoskeletal components revealed that induction of mechanical memory is actin-dependent in CFs and microtubule-dependent in CMs. Prevention of α-tubulin detyrosination blocks both induction and maintenance of mechanical memory in CMs. Overall, these results emphasize the distinct temporal dynamics of mechanical memory in CMs and CFs with different cytoskeletal mediators responsible for inducing and maintaining the stiffness activated phenotype. Due to its flexibility, this model is broadly applicable to future studies interrogating mechanotransduction and mechanical memory in the heart and might inform strategies for attenuating the impact of load-dependent pathology and excess myocardial stiffness.
Abstract P2075: The Effects Of Maternal Psychosocial Stress During Pregnancy On Offspring Heart Health
Authors: Sturgill Sarah L , Ziolo Mark T , Taryn Lackritz E
Published: October 2023

Maternal health has a substantial effect on offspring well-being and health. A significant contributing factor to maternal health is psychosocial stress. Psychosocial stress results from adverse experiences in everyday life and the inability to cope with them, resulting in anxiety and depression. Unfortunately, it is estimated that ~75% of mothers will suffer a stressful life event the year their infants are born, and upwards of 20% suffer depression. These already high levels of psychosocial stress have been exacerbated by the COVID-19 pandemic. However, it is not known if maternal psychosocial stress during pregnancy affects offspring heart health. Thus, the
purpose
of this study was to investigate the effects of maternal psychosocial stress during pregnancy on the heart health of offspring. We
hypothesize
that maternal psychosocial stress during pregnancy will have an adverse effect on offspring heart health.

Methods:
We used a well-established model of maternal psychosocial stress during pregnancy- restraint stress. Pregnant dams were stressed by placing them in a 50 mL conical tube (with air holes) for 2 hours / day on gestational days 10-17. Offspring heart health was assessed via echocardiography (Vevo 3100, MX550D transducer).

Results:
As early as one week of age (equivalent age of infancy in humans), we observed that offspring born to stressed dams exhibited decreased left ventricular fractional shortening. Further analysis using speckle tracking echocardiography was performed on offspring at 4 (equivalent age of juveniles in humans) and 12 (equivalent age of young adult in humans) weeks of age. Our data indicate that offspring from stressed dams had decreased heart performance (measured as stroke volume), remodeling observed as hypertrophy (measured as left ventricular posterior wall thickness at diastole), and diastolic dysfunction (measured as E’/A’).

Conclusion:
Our data indicate maternal psychosocial stress during pregnancy results in blunted left ventricular contractile and diastolic dysfunction in offspring, as well as displays adverse remodeling (i.e., hypertrophy) during infancy and through young adulthood. These data indicate that maternal psychosocial stress during pregnancy is detrimental to offspring heart health.
Abstract P2106: Transforming Growth Factor β-dependent Regulation Of Cardiomyocyte Maturation
Authors: Minerath Rachel A , Grimes Kelly , Kasam Rajesh K , Sargent Michelle , York Allen , Kuwabara Yasuhide , Saviola Anthony , Alfieri Christina , Hansen Kirk , Yutzey Katherine E , Molkentin Jeff D
Published: October 2023

Transforming growth factor β (TGFβ) is a secreted growth factor that is sequestered to the ECM as a latent complex and released in response to cardiac stress. Once released, TGFβ is required for conversion of quiescent fibroblasts to myofibroblasts that support cardiac repair and fibrosis through production of ECM components. While these functional features of TGFβ are well known, the fibroblast-independent effector functions of TGFβ in the heart are unknown. We hypothesized that TGFβ is a critical ECM-cell cross talk mediator in the heart whereby cardiomyocyte -generated TGFβ communicates with fibroblasts to program the composition and stability of the ECM in a reinforcing feedback network. We found that deletion of TGFβ ligands (
Tgfb1
,
Tgfb2
, and
Tgfb3
) from cardiomyocytes with an α-MHC-Cre transgene (

Tgfb123
fl/fl

-αMHC-Cre
), but not a fibroblast-specific targeted allele (

Tgfb123
fl/fl

-Tcf21-MCM
), results in cardiac dysfunction. These results suggest that cardiomyocytes (CMs) are the predominant source of TGFβ in the heart at baseline and that perhaps celltypes beyond the fibroblast were also involved. Interestingly, fibroblast numbers and ECM deposition are increased in

Tgfb123
fl/fl

-αMHC-Cre
hearts and with acute injury hearts from these mice produce shorter, disorganized collagen fibers. Gene expression profiling of

Tgfb123
fl/fl

-αMHC-Cre
hearts at 6 weeks of age demonstrates defective cardiomyocyte maturation. Indeed,

Tgfb123
fl/fl

-αMHC-Cre
hearts are dysmorphic at neonatal day 1 and cardiomyocytes from these hearts remain in cell cycle for a longer duration with greater levels of mononucleation. However, deleting TGFβ receptors I/II or Smad2/3 from CMs does not recapitulate this phenotype suggesting that TGFβ is not driving CM maturation in an autocrine manner. Instead, these findings indicate that TGFβ functions as a critical cross-talk mediator to fibroblasts to establish proper ECM organization and content that drives CM maturation.
Abstract P2117: Hypoadiponectinemia-induced Epigenetic Downregulation Of Endogenous Antioxidants Exacerbates Cardiac Ischemia-reperfusion Injury In Diabetic Mice
Authors: Zhao Jianli , Meng Zhijun , Wu Yalin , liu caihong , Gao Erhe , Koch Walter J , Zhao Fujie , Lopez Bernard L , Christopher Theodore , Zhang Jianyi J , Ma Xinliang L , Wang Yajing
Published: October 2023

Diabetes worsen myocardial ischemic/reperfusion (MI/R) injury, but the underlying mechanism is still not fully understood. Epigenetics regulation is markedly involved in the process. Adiponectin (APN), a cardioprotective adipokine, is suppressed by diabetes, and dysregulated miRNAs are implicated in disease development. However, it is unclear whether hypoadiponectinemia alters cardiac miRNA profile, contributing to diabetic heart injury. Wild-type (WT) and APN knockout (APN-KO) mice were subjected to MI/R and cardiac microRNA profile were determined. Comparing those miRNAs that increased in APN-KO mice following MI/R, miR-449b was most significantly upregulated (3.98-fold over WT mice). Administering miR-449b mimic increased apoptosis, enlarged infarct size, and impaired cardiac function in WT mice. In contrast, anti-miR-449b decreased apoptosis, reduced infarct size, and improved cardiac function in APN-KO mice. In depth Bioinformatic analysis revealed oxidative stress as the top pathway regulated by miR449b. Venn analysis followed by luciferase assay identified Nrf-1 and Ucp3 as the two most important miR-449b targets. Administrating anti-miR-449b to APN-KO mice attenuated MI/R-stimulated superoxide overproduction. High glucose/high lipid in vitro treatment simulated ischemia/reperfusion upregulated miR-449b, leading to inhibition of Nrf-1 and Ucp3. These pathological effects were attenuated by anti-miR-449b or Nrf-1 overexpression. Clinically relevant cohorh study in high-fat diet (HFD)-induced diabetic mice followed with MI/R identified the copied pheonotype. Administration of anti-miR-449b or APN preserved cardiac Nrf-1 expression, reduced cardiac oxidative stress, decreased apoptosis and infarct size, and improved cardiac function. In conclusion, the study shows that hypoadiponectinemia upregulates miR-449b, suppressing Nrf-1/Ucp3 expression, which leads to oxidative stress and worsens MI/R injury. The APN/miR-449b/oxidative stress pathway could potentially be a therapeutic target for diabetic MI/R injury.
Abstract P2158:
In Vivo
Dissection Of A CRISPR/Cas9-Mediated Precise Genome Editing Mechanism
Authors: Dey Sanchita S , VanDusen Nathan J
Published: October 2023

Genomic sequencing has uncovered many genetic variations that interfere with development and function. Our inability to make precise and permanent genome edits limits our ability to treat genetic disorders. However, CRISPR/Cas9 has been demonstrated to be a potent tool for genome editing, with enormous therapeutic potential. Furthermore, the use of adeno-associated virus (AAV) to deliver CRISPR/Cas9 components, as well as a donor template for homology directed repair (HDR; AAV-HDR), has shown impressive editing efficiencies, particularly
in vitro
. In comparison,
in vivo
studies have shown more modest editing efficiencies, with large variation between target loci. Thus, to employ this strategy therapeutically it is imperative to establish a robust understanding of the underlying mechanism, which can then be exploited to improve efficiency. Thus far, the absence of high-throughput tools that enable systematic analysis has hindered efforts to explore and enhance AAV-HDR efficiency. Here we employ two novel high-throughput methodologies for measuring
in vivo
AAV-HDR editing efficiency in cardiomyocytes of Cas9-expressing mice. In our first approach we analyze the locus-dependent variability of AAV-HDR efficiency, with the goal of identifying the genomic characteristics of high-efficiency loci. In the second approach, we utilize a high-throughput pooled CRISPR KO screen to identify the DNA-repair factors required for efficient gene editing. This approach has uncovered several novel regulators of cardiac AAV-HDR, which we are currently working on characterizing. Collectively these studies will provide insights into the molecular mechanism of AAV-HDR, and we anticipate that these advances will promote development of AAV-HDR based therapies.
Abstract P2176: Development Of Integrated Transparent Transgenic Zebrafish Modeling System For Experimental Cardio-oncology Therapeutics
Authors: Rajpurohit Surendra K , Ouellette Logan , Balyan Vaani , Bhatt Keshu , Van Derhei Aiden , Aikhionbare Karen , Chen Raymond , Gossman Taitum , Nasanally Sai , Lokeshwar Balakrishna , Arora Vishal
Published: October 2023

The zebrafish model has provided an excellent platform to study the genetic and molecular approach of basic and translational cardiac research. Zebrafish heart cells are similar to human heart cells at the molecular level and determine the function of genes that control cardiac function and dysfunction. In zebrafish, myl7 is myosin light chain-7 gene and identified as a regulatory gene of heart orthologs to human MYL7. In vertebrates, including zebrafish, murine and human systems, the in-vivo spatial resolution is limited due to the normal opacification of skin and subdermal structures. For in-vivo imaging the skin transparency is primary requirement and to maintain the transparency, blocking the pigmentation needs to maintain. Chemical inhibition treatment is temporary and possible till the organism treated with the chemical inhibitor agent. Zebrafish casper mutant maintain transparency throughout the life and serve as ideal combination of sensitivity and resolution for in-vivo stem cell analyses and in-vivo imaging. To maintain the skin transparency, in this study, we have developed transparent transgenic zebrafish model and established of time lapse in-vivo confocal microscopy to study of cellular phenotype/pathologies of the cardiomyocytes to quantify changes in cardiomyocyte morphology and function overtime by comparing control and cardiac injury by generating casper/ myl7:RFP; annexin-5:YFP transgenic zebrafish. In the field of cardio-oncology, we are developing in-vivo transparent transgenic cellular phenotype model of zebrafish to study of the cell death process (Annexin5) and Inflammatory activity (NFkB) in the microglia, and the resident macrophage of the brain and cardiomyocytes) homozygous
casper/ myl7:RFP; annexin-5:YFP/NF-kB:GFP/mpeg1:mcherryFP
transgenic strain zebrafish. By using this novel model our lab establishing the role of NFkB and microglia function under normal healthy status and in glioblastomas Our strategist approach to yield crucial new insights into in-vivo cardiomyocyte imaging by confocal microscopy to observe and track the cell death pattern and cardio inflammatory pathways in cardiomyocyte and develop novel therapeutic approaches to treat cardio inflammatory pathology and glioblastoma
Abstract P2182: Cardioprotective Effect Of APE1/Ref-1 In Doxorubicin-induced Cardiotoxicity
Authors: An Soo Yeon , Seo Hee Jeong , Jin Seon-Ah , Jeong Jin-Ok
Published: October 2023

Doxorubicin is a potent anthracycline anticancer drug widely used to treat various malignancies. Unfortunately, it can cause negative side effects such as cardiomyopathy. Apurinic apyrimidinic endonuclease 1/Redox factor-1 (APE1/Ref-1) is a versatile protein capable of being secreted from cells, but the role of secreted APE1/Ref-1 in doxorubicin-induced cardiomyopathy remains unknown. In this study, we used Adenoviral preprotrypsin leading sequence APE1/Ref-1 (Ad-PPTLS-APE1/Ref-1) to overexpress secreted APE1/Ref-1 in cells and mice through transfection. We aimed to investigate the potential role of secreted APE1/Ref-1 in preventing doxorubicin-induced cardiomyopathy, a serious side effect associated with the use of this widely used anticancer drug. Our findings reveal that exposure to secreted APE1/Ref-1 significantly decreases NT-proBNP levels, a known marker for cardiomyocyte injury, in doxorubicin-treated H9C2 cells. Furthermore, secreted APE1/Ref-1 was found to reduce the severity of cardiomyocyte injury and apoptosis both in vitro and in vivo models of doxorubicin cardiotoxicity. These cardioprotective effects were mediated through the inhibition of the p53 signaling pathway and the enhancement of cell viability in doxorubicin-treated cardiomyocytes.Our study provides evidence that secreted APE1/Ref-1 has the potential to attenuate oxidative stress and inhibit doxorubicin-induced apoptosis both in vitro and in vivo. Based on our results, supplementing secreted APE1/Ref-1 to patients at high risk of doxorubicin-induced cardiotoxicity could be a promising strategy for reducing the harmful effects of doxorubicin. Further studies are needed to fully understand the mechanism by which APE1/Ref-1 protects against doxorubicin-induced cardiotoxicity and to determine if this finding can be translated to human patients.
Abstract P3026: Multimodal Imaging To Identify Pre-signals For Acute Aortic Dissection By Using Marfan Syndrome Model Mouse
Authors: SUGIYAMA KAORI , Ando Masahiro , Matsunaga Hiroko , Wagatsuma Ryota , Uchino Haruki , Kimura Kenichi , Schenke-layland Katja , Arita Makoto , Yanagisawa Hiromi , Takeyama Haruko
Published: October 2023

Background:
Marfan syndrome (MFS) is a genetic disorder caused by mutations in the gene encoding theextracellular matrix Fibrillin-1 (FBN1), resulting in systemic connective tissue abnormalities. The mostcommon life-threatening conditions are aortic rupture and/or dissection. Aortic dissections show a highmortality rate, but the details of molecular triggers are unknown, particularly in acute aortic dissection(AAD). Raman spectroscopy is a technique for analyzing molecular structures from Raman spectra. It hasrecently attracted attention in the biology field because it can non-invasively distinguish between diseaseand non-disease areas.

Objectives:
This study aimed to identify the signals that induce changes in the aortic wall prior to acuteaortic dissection using genetic analysis and Raman spectroscopy with preservation of positional information.

Methods:
An acute aortic dissection model was created by using MFS model mice (Fbn1
mgR/mgR
) byadministration of angiotensin II (AngII) via osmotic pump (Fbn1
mgR/mgR
-AAD). Aortas from wild-type(WT),
Fbn1

mgR/mgR

and
Fbn1

mgR/mgR

-AAD mice were harvested, and frozen sections were prepared. Thevascular wall of each mouse was assessed histologically, followed by spatial transcriptomic analysis andRaman microscopy measurements. Multivariate data analysis was performed on the obtained Ramanspectra. Mass spectrometry (MS) imaging was utilized for lipidome analysis.

Results:
Label-free imaging of extracellular substrates (collagen fibres, elastic fibres, versican, andaggrecan), lipids, and cells was produced in WT,
Fbn1

mgR/mgR

and
Fbn1

mgR/mgR

-
AAD aortic tissue. Spatialtranscriptomic analysis identified differentially expressed genes among these aortas. We identified markedlipid accumulations in the acute aortic dissection models with Raman and MS imaging.
Abstract P3047: Alternative Splicing Networks During Postnatal Mouse Atria Development
Authors: Gentile Gabrielle , Blue Eric , Giudice Jimena
Published: October 2023

The heart is remodeled during the first four postnatal weeks to meet the increasing physical demands of the body. One driver of these changes is alternative splicing. Alternative splicing is an RNA processing mechanism that enables one gene to generate more than one transcript. Alternative splicing is ubiquitous, occurring in over 95% of human genes. The heart exhibits one of the most tissue specific and highly conserved alternative splicing programs, and mis-splicing is a hallmark of cardiovascular diseases. We previously identified extensive splicing changes during postnatal development in mouse ventricles; however, we still lack a molecular landscape of the developing atria. Here, we performed deep RNA-sequencing on mouse atria at four postnatal ages: P4.5, P10, P28, P90. We observed substantial changes in events that undergo alternative splicing of a single exon. These transitions are concentrated in the first four weeks (503 events between P4.5-P28; 83 events between P28-P90), suggesting they are functionally important for this developmental window. Gene Ontology (GO) analysis of the 503 events revealed pathways related to trafficking, intracellular architecture, and cardiac conduction. Notably, 287 (57%) of the alternative exons have sizes that are a multiple of three (in-frame), and the associated genes encode proteins that function in cell adhesion and extracellular matrix organization, while out-of-frame exons are present in genes encoding cell cycle proteins and transcription factors. We hypothesize that alternative splicing of in-frame exons imparts a novel functional role to the encoded protein, while splicing out-of-frame exons induces transcript instability and degradation. Alternative splicing is mainly regulated by RNA binding proteins (RBPs). We identified one upregulated RBP (RBFOX1) and two downregulated RBPs (FMR1, TIA1) between P4.5-P28, suggesting that these RBPs coordinate alternative splicing networks to promote atria development. Our efforts are focused on elucidating the mechanism by which these RBPs control alternative splicing in the developing atria as well as the functional consequences associated with each event. These findings will be fundamental to our understanding of cardiac development and disease.
Abstract P3065: Mechanistic Insights Into Cardiac Dysfunction And Remodeling Associated With Invasive Pneumococcal Pathogenesis
Authors: Sultan Tousif , Minassian Daniel , Umbarkar Prachi , He Chao , Xie Min , Sethu Palaniappan , Orihuela Carlos J , Lal Hind
Published: October 2023

Background:
One-in-four adults hospitalized for community-acquired pneumonia (CAP) experiences a major adverse cardiac event (MACE). Pneumonia survivors, in particular those who experienced invasive pneumococcal disease, are also at elevated risk for MACE in convalescence. Although
Streptococcus pneumoniae
(
Spn
)-mediated cardiac dysfunction is a well-established clinical problem, the mechanisms by which pneumonia impacts the long-term risk of MACE are poorly understood.

Methods and Results:
Each mouse was infected with 1 x 10
4
CFU of
Spn
, to model invasive pneumococcal disease (IPD). At 30 hours post-infection, mice were administered Ampicillin (50mg/kg) every 12 hours for 3 days. After that, animals were given Ampicillin (1mg/ml) ad libitum in drinking water for 4 days. Three weeks post-infection phase,
Spn
-rescued animals’ demonstrated marked cardiac dysfunction as reflected by significantly reduced ejection fraction (EF), and fractional shortening (FS). Cardiac injury was confirmed to have occurred by measurement of increased expression of ANP, BNP (q-PCR), and levels of serum Troponin-I as measured by ELISA. Trichrome-stained heart sections demonstrated fibrotic remodeling in the
Spn
-rescued animals. To gain insight into mechanisms, we analyzed whether
Spn
alters cardiac immune cell infiltration and pro-inflammatory S100A8/A9-TLR-NLRP3-IL-1β signaling axis
in vivo
. Indeed,
Spn
-rescued (with antibiotic) animals showed an increased percentage of TLR-2/4+, NLRP3+, and phosphor-NFkB+ myeloid/neutrophils in the heart. Importantly, these findings were corroborated using human blood samples collected from patients with IPD. Pharmacological blocking of S100A8/A9-TLR-NLRP3-IL-1β signaling axis by S100A9 inhibitor largely reduced
Spn
-induced detrimental cardiac pathologies in convalescence.

Conclusion:
Our results suggest chronic activation of the pro-inflammatory S100A8/A9-TLR2/4-NLRP3-IL-1β pathway in
Spn
-infected hearts that persists even after antibiotic clearance of bacterial infection. Targeting the S100A8/9-NLRP3-IL-1β signaling circuit reduced chronic inflammation and fibrosis in our mice model and therefore is potentially a novel strategy to mitigate the Spn-mediated cardiac dysfunction.
Abstract P3089: Selective Activation Of Cardiac Macrophages To Study Injury And Repair
Authors: Kasam Rajesh K , Grimes Kelly M , Johansen Anne Katrine Z , Vagnozzi Ronald J , Molkentin Jeffery D
Published: October 2023

Objective:
Pathophysiological inflammatory responses play an important role during ischemia-mediated heart injury and remodeling, where monocytes and macrophages appear to be critically involved. Cardiac macrophages are generally classified as a recruited, monocyte-derived subset that is Ccr2
+
and a tissue resident subset that is typically Ccr2
-
. Recent studies have shown that these two subsets have divergent functions with Ccr2
-
macrophages being pro-regenerative while Ccr2
+
macrophages are pro-inflammatory. Here we selectively manipulated the content and/or activation of these two macrophage subsets in the heart.

Methods and Results:
To study the function of Ccr2
+
cardiac macrophages in the heart, we have overexpressed Ccl-2 (Mcp-1) ligand specifically in cardiomyocytes by using adeno-associated virus serotype-9 (AAV9) mediated gene transfer. Postnatal day 4 (p4) Ccr2
RFP/+
pups were injected with either AAV9-Ccl-2 or AAV9-control. At two months of age, we observed increased Ccl-2 transcripts and protein expression in the heart as measured by qRT-PCR and ELISA, respectively. By quantifying immune cells using flow cytometry, we observed increased infiltration of Ccr2
+
macrophages (CD45
+
CD11b
+
CD64
+
) in the hearts of AAV9-Ccl2 injected mice compared to controls. However, echocardiography analysis revealed no change in cardiac function in adult Ccl-2 overexpressed mice. Moreover, the recruitment of Ccr2
+
macrophages into the myocardium did not cause fibroblast expansion or activation.

Conclusion:
Our results show that cardiomyocyte-specific overexpression of Ccl-2 induces profound infiltration of Ccr2
+
macrophages into the myocardium but this did not alter cardiac function or cause fibrosis at two or six months of age.
Abstract P3167: Identification Of Macrophage-specific Markers On Extracellular Vesicles In A Transgenic Mouse Model And Humans
Authors: Carnel-Amar Natacha , Saftics Andras , Abu-Elreich Sarah , Spanos Michail , Chatterjee Emeli , Limpitikul Worawan , Ghiran Ionita , Gould Stephen , Raffai Robert , Van Keuren-Jensen kendall , Jovanovic-Talisman Tijana , Das Saumya
Published: October 2023

Extracellular Vesicles (EVs) are key mediators of intercellular communication. Macrophages, a prominent type of immune cells, are known to release EVs with distinct contents and functions. The ExoMap mice model provides a platform to investigate tissue-specific EV subpopulations and the complexity of EV heterogeneity. CD169 and CD68 markers are specific to macrophages and have been associated with immune diseases, including Sarcoidosis. Double Transgenic vav-cre/ExoMap mice and appropriate controls were used to assay for immune cell derived EVs. Expression of cre in the vav-expressing cells, leads to expression of humanized CD81 (hsCD81) and mNeonGreen in the immune cells and in their EVs. Size Exclusion Column and immuno-pulldown (IP) with anti-hsCD81 or anti-CD169/CD68 antibodies were used to isolate macrophage-specific EVs. 3 miRNAs (mir223, mir374c, mir412) specific to macrophages were tested using Droplet Digital PCR (ddPCR). To design our human pilot study, we chose patients with active sarcoidosis (n=10), latent sarcoidosis (n=10), healthy controls (n=10) to compare the level of expression of specific immune markers in their blood. Bone marrow cells prepared from vav-cre/ExoMap mice were found to express mNeon Green and hsCD81. IP combined with vesicle flow cytometry defined the presence of hsCD81/mNeonGreen EVs in the plasma of double transgenic mice. Expression of CD68 and CD169 was robust on the EVs isolated using IP with hsCD81 antibodies from Double Transgenic mice. These results are consistent with the immune specificity of the vav-cre expression. Using human pooled plasma, we detected expression of CD68 on the EVs isolated using IP with CD169 antibody, suggesting the presence of both markers on macrophage specific EVs. Our findings support these markers as conserved between mouse and human samples, indicating their potential as universal markers of macrophage-derived EVs. Ongoing experiments aim to comprehensively characterize individual macrophage-derived EVs using the quantitative single molecule localization microscopy (qSMLM). Together, our findings provide a valuable resource for future studies of macrophage-EV communication and could serve in the development of EV-based diagnostic and therapeutic strategies.
EUROPEAN UNION CARBON BORDER ADJUSTMENT MECHANISM: A SWOT ANALYSIS FOR TÜRKİYE
Authors: ÖZEKAN Damla
Published: October 2023

The European Union’s (EU) proposed Carbon Border Adjustment Mechanism (CBAM) has gained attention as a potential policy tool to address carbon leakage and promote climate policy alignment in international trade. This paper examines the implications of the EU CBAM for Türkiye and conducts a SWOT analysis to evaluate Türkiye’s strengths, weaknesses, opportunities and threats in the context of CBAM implementation. In the paper, firstly EU’s environmental policy, European Green Deal (EGD) and CBAM are defined and examined in historical perspective. Secondly Türkiye’s environmental policy on behalf of CBAM is explained. Then, the effects of EGD and CBAM on Türkiye’s foreign trade is discussed. The last part is devoted to SWOT analysis. The analysis begins by exploring Türkiye’s strengths including its domestic climate policies and diverse economy. The analysis also highlights the weaknesses that could challenge Türkiye’s competitiveness under the CBAM. It identifies opportunities within the CBAM framework including incentives for green investments and collaboration for increased market access and also acknowledges potential threats such as trade disputes.
Reinstatement of Sapium sceleratum (Euphorbiaceae), an endemic species from Northeast Brazil, and new circumscription of Sapium argutum
Authors: CORDEIRO WESLEY PATRÍCIO FREIRE DE SÁ , MELO ANDRÉ LAURÊNIO DE , ATHIÊ-SOUZA SARAH MARIA , ESSER HANS-JOACHIM , SALES MARGARETH FERREIRA DE
Published: October 2023

This paper includes the reinstatement of Sapium sceleratum with one new synonym (Sapium cicatricosum). In addition, a new circumscription is presented for Sapium argutum and the second-step lectotypification of the name Excoecaria arguta, as well as an identification key for the species of Sapium from North-east Brazil, illustrations and comments on geographic distribution and habitat of S. argutum and S. sceleratum.
Inhibition of Transverse Aortic Constriction Induced Myocardial Hypertrophy by Knocking out the Target of the Early Growth Response-1 to Inhibit Apoptosis and Autophagy
Authors: Wu Huiping , Li Jie , Xia Tianhe , He Yue’e , Wu Tingting , Wang Zhenquan , Song Shiyang , Chu Maoping , Rong Xing
Published: October 2023

Myocardial hypertrophy, a significant contributor to the development of heart failure, continues to be prevalent. Early growth response-1 (EGR-1) is closely linked to the development of diverse myocardial conditions. The target of EGR1 (TOE1) is a critical factor in myocardial hypertrophy,
but its regulatory function remains unclear. Myocardial cell injury was induced by angiotensin II. TOE1 knockout mice and cells were generated to investigate its impact on myocardial hypertrophy. TUNEL staining was employed to assess cell apoptosis. Furthermore, western blotting and qRT-PCR
were performed to measure the expression of target genes. The results revealed that knockout of TOE1 effectively inhibited myocardial hypertrophy and injury caused by transverse aortic constriction. In vivo experiments demonstrated that TOE1 knockout improved myocardial function and
suppressed inflammatory factors, oxidative stress, apoptosis, and autophagy levels. In vitro, TOE1 knockout suppressed cell apoptosis, mitochondrial damage, and the intensity of reactive oxygen species. Additionally, it inhibited the expression of apoptosis- and autophagy-related genes.
These findings introduce a promising avenue for preventing and treating myocardial hypertrophy.
The Role of Gold Nanorods in Detecting Circulating microRNAs as Biomarkers in Liver Diseases
Authors: Yang Sheng , Liu Chong , Ji Xiaowen , Chen Xuemin , Wang Yun , Tao Rong
Published: October 2023

Liver disease is one of the most significant diseases in humans. Various factors can lead to liver degeneration and necrosis, fatty liver, cirrhosis, etc. This study evaluated the diagnostic value of circulating miRNAs in liver injury and examined the role of gold nanorods (AuNRs) in
the detection of biological indicators. We collected data from patients with liver injury in three groups. Healthy volunteers were used as the control group, and the levels of serum markers of liver injury were detected in all participants. The circulating miRNAs associated with liver injury
were screened by searching the database, and circulating miRNAs were detected by qRT-PCR. The sensitivity and specificity of circulating miRNAs in diagnosing liver injury were analyzed using receiver operating characteristic curve. AuNRs were prepared to evaluate the concentrations of glucose,
cholesterol, uric acid, and alkaline phosphatase (ALP), which were compared with the test results of the kit. We found that the levels of alanine aminotransferase and aspartate aminotransferase changed significantly in patients with drug-induced liver injury (DILI). Total bilirubin levels
increased significantly in patients with both non-alcoholic steatohepatitis (NASH) and DILI, but difference between the two groups was insignificant. MDH and adenosine deaminase levels increased in patients with liver injury, whereas paraoxonase-1 levels decreased, which can be combined with
alanine aminotransferase levels to diagnose patients with liver injury. Compared with the control group, circulating miR-106-5p, miR-194-5p, and miR-29b-3p were specifically expressed in tissues with liver injury: miR-106-5p expression was lowest in NASH, miR-194-5p expression was highest
in chronic hepatitis B (CHB) and NASH, and miR-29b-3p expression was highest in CHB. Receiver operating characteristic curve analysis revealed that miR-106-5p, miR-194-5p, and miR-29b-3p can be used to diagnose patients with NASH, DILI, and CHB, respectively. The combination of miR-194-5p
and miR-29b-3p showed good diagnostic ability. The results of biological indicator detection using AuNRs were similar to those detected by the kit. Therefore, AuNRs are a promising candidate for biological detection. This study validated the diagnostic value of circulating miRNAs in liver
disease and the application of AuNRs in the detection of biological indicators, providing more theoretical basis for the role of circulating miRNAs in disease prevention and diagnosis.