bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract GS.09: Interleukin 13 Signaling To Macrophages Promotes Recovery After Myocardial Infarction In Mice
Authors: Alvarez-Argote Santiago , Omeara Caitlin
Published: October 2023

Introduction:
There is great interest in identifying signaling pathways that promote pro-reparative macrophage phenotypes in the heart. Considering the remarkable cardiac healing potential of neonatal mice, we employ this model to identify pro-reparative mechanisms that can be applied to promote cardiac repair in adults after myocardial infarction (MI). We show that IL13/IL4Rα signaling specifically on macrophages mediates neonatal heart regeneration and activation of this pathway in adult mice promotes cardiac functional recovery in post-MI.

Results:
We developed IL4Rα
fl/fl
;CX3CR1
Cre
mice (IL4Rα
MacKO
) to delete IL4Rα specifically in macrophages. Neonatal IL4Rα
MacKO
and control mice were subjected to MI on postnatal day 1 (P1). Following injury, IL4Rα
MacKO
mice had significantly larger scars at 8 days post injury (dpi) and lower ejection fraction and capillary density at 21 dpi. Thus, macrophage IL4Rα expression is critical for cardiac functional recovery following injury in neonates. Using fluorescent reporter mice, we found that IL13, a ligand for IL4Rα, is produced almost exclusively by type 2 innate lymphoid cells in the neonatal heart. Interestingly, we didn’t find any cellular source of IL13 in the adult mouse heart. Considering the decline in cardiac production of IL13 from neonatal to adult stages, we postulated that reactivation of IL4Rα in the adult heart would improve cardiac healing post MI in adults. Indeed, daily IL13 administration significantly improved cardiac function post MI in adult mice, and this improvement was mediated entirely by IL4Rα expression on macrophages as IL4Rα
MacKO
mice showed no functional improvement with IL13 administration. Intriguingly, while IL13 administration to control mice improved cardiac function, administration to IL4Rα
MacKO
mice further exacerbated post MI mortality and scar size compared to animals receiving PBS, suggesting multiple cardiac cell types respond to IL13 resulting in beneficial or detrimental outcomes.

Conclusions:
Our data elucidates a strongly pro-reparative role for IL13 signaling directly to macrophages. While this signaling pathway is active in neonatal stages, we demonstrate that re-activation by IL13 administration is required to promote cardiac repair in adults.
Abstract GS.102: Cd206
+
Il-4rα
+
Macrophages Are Drivers Of Adverse Cardiac Remodeling In Ischemic Cardiomyopathy
Authors: Wang Qiongxin , Ismahil Ameen , Rokosh Gregg , Hamid Tariq , Zhou Guihua , Zhu Yujie , Pogwizd Steven M , Prabhu Sumanth D
Published: October 2023

Background:
Cardiac macrophages are significantly expanded in chronic heart failure (HF); however, their role is unclear. We tested the hypothesis that CD206
+
macrophages expressing interleukin (IL)-4Rα in the failing heart are key drivers of adverse left ventricular (LV) remodeling and ischemic cardiomyopathy.

Methods and Results:
Adult male C57BL/6 (WT) mice underwent non-reperfused myocardial infarction (MI) to induce HF, or sham operation, and cardiac macrophages were profiled using flow cytometry. As compared to sham hearts, both the frequency and absolute number of CD206
+
macrophages steadily increased in post-MI hearts during the progression of LV remodeling and HF, such that at 8 w post-MI they comprised ~85% of macrophages. These macrophages were exclusively proliferative and predominantly C-C motif chemokine receptor (CCR2)
-
with nearly half expressing IL-4Rα, and correlated with LV dysfunction and fibrosis. IL-4-polarized bone marrow derived CD206
+
macrophages exhibited marked upregulation of found in inflammatory zone (FIZZ)1 and induced FIZZ1-dependent myofibroblast differentiation of cardiac mesenchymal stem cells (cMSCs), in part related to DLL-4/Jagged1-Notch1 signaling. Intramyocardial adoptive transfer of M[IL-4], but not M[IL-10], macrophages induced progressive LV dilation and dysfunction and augmented cardiac fibrosis over 4 weeks in naïve mice.
In vivo
IL-4Rα gene silencing in mice with established HF effectively depleted cardiac CD206
+
IL-4Rα
+
macrophages and induced reverse LV remodeling, improving fibrosis, neovascularization, and dysfunction, and suppressing local and systemic inflammation. Lastly, alternatively activated CD206
+
and CD163
+
macrophages were significantly expanded in human failing hearts and correlated with cardiac fibrosis, with the majority of CD163
+
macrophages expressing IL-4Rα and FIZZ3, the human homolog of FIZZ1.

Conclusion:
CD206
+
IL-4Rα
+
macrophages proliferate and expand in the failing heart and are key mediators of pathological remodeling and fibrosis, in part through the secretion of FIZZ1. Inhibition of CD206
+
macrophage IL-4Rα signaling may be a fruitful therapeutic approach for alleviating LV remodeling in HF.
Abstract P1016: Single Cell Transcriptomic Analysis Of Tafazzin Knockout Mice For Pathway And Therapeutic Discovery In Barth Syndrome
Authors: Perera Gayani , Power Liam , Larson Amy , Codden Christina , Awata Junya , Batorsky Rebecca , Strathdee Douglas , Chin Michael T
Published: October 2023

Barth Syndrome, a rare X-linked disorder affecting 1:300,000 live births, results from defects in Tafazzin, an acyltransferase that remodels cardiolipin and is essential for mitochondrial respiration. Barth Syndrome patients develop cardiomyopathy, muscular hypotonia and cyclic neutropenia during childhood, rarely surviving to middle age. At present, no effective therapy exists and downstream transcriptional effects of Tafazzin dysfunction are incompletely understood. To identify novel, cell-specific pathological pathways downstream of Tafazzin that mediate heart dysfunction, we performed snRNA-seq on mouse hearts deficient in Tafazzin. We generated libraries from four WT and four KO mouse hearts (10X Genomics Chromium) and performed next generation sequencing (Illumina NovaSeq S4), demultiplexing (CellRanger), doublet reduction (DoubletFinder), ambient RNA removal (SoupX), sample integration (Harmony), optimal clustering (ChooseR/Seurat), determined differentially expressed genes (DEGs; DESEQ2), and inferred a predicted cell-cell communication network (scLR). Surprisingly, DEGs were distributed heterogeneously, with cardiomyocytes, adipocytes, endothelial cells, fibroblasts and macrophages exhibiting the greatest number of DEGs between each genotype, while lymphocytes, lymphatic endothelium, pericytes, smooth muscle cells and mesothelial cells were mostly unaffected. Gene Ontology analysis showed cell specific effects such as altered amino acid and carbohydrate metabolism plus altered ion channel function in cardiomyocytes and common metabolic perturbations across all affected cell types. Analysis of Ligand-Receptor pair expression, to infer intercellular communication patterns, revealed WT adipocytes form the densest intercellular communication network, followed by WT cardiomyocytes. In the Tafazzin KO hearts, adipocyte significant intercellullar communication ceases, primarily through loss of PDGFRB interactions, while cardiomyocytes remain largely unperturbed. These findings suggest that adipocyte mitochondria may be most sensitive to mitochondrial Tafazzin deficiency and rescuing adipocyte mitochondrial dysfunction may provide therapeutic benefit in Barth Syndrome patients.
Abstract P1018: Peptidyl-prolyl Cis-trans Isomerase-like 4 Is A Novel Regulator Of Smooth Muscle Cell Remodeling
Authors: Gultekin Batur , Yalcin Kanat , Antonios Joseph , Miyagishima Danielle , Erson Omay Zeynep , Barak Tanyeri , Gunel Murat
Published: October 2023

Introduction:
Smooth muscle cell (SMC) remodeling is a complex and multifaceted process that plays an essential role in the maintenance and function of various organ systems in the body. In response to physiological and pathological stimuli, smooth muscle cells exhibit remarkable plasticity, undergoing morphological and functional changes that can impact vascular tone. Dysregulation of SMC remodeling can contribute to the development and progression of numerous pathological conditions, such as hypertension and atherosclerosis. However, the role of SMC remodeling in intracranial aneurysm (IA) formation has yet to be explored.

Objective-Method:
In this study, we aimed to elucidate the role of PPIL4, an IA-implicated gene, in SMC remodeling using a targeted experimental approach. We performed immunostaining and bulk RNA sequencing (RNA-seq) in human primary brain vascular smooth muscle cells (HPBVSMC) treated with lentiviral shRNA and open-reading-frame (ORF) particles to individually downregulate and overexpress PPIL4, respectively.

Results:
We found that PPIL4 downregulation results in significant upregulation of the genes encoding contractile proteins, including ACTA2, CNN1, TNNT1, and TNNT2, in HPBVSMC and leads to a significantly increased cell size, favoring a contractile SMC phenotype. Conversely, overexpressing PPIL4 using ORF-lentiviral particles caused downregulation of contractile proteins, increased expression of MMP1 and MMP3, and promoted a synthetic phenotype in SMCs. Furthermore, PPIL4 overexpression caused cell elongation and spindle-like cell formation in HPBVSMC.

Conclusion:
In conclusion, our findings highlight the critical role of PPIL4 in modulating SMC remodeling, suggesting a potential mechanistic link between PPIL4 dysregulation, SMC remodeling, and IA formation, which warrants further investigation.
Abstract P1026: Structural And Biochemical Effects Of Missense MyBP-C Mutations On The Myofilament In Human Hypertrophic Cardiomyopathy
Authors: McAllister Christopher M , Jani Vivek , Nissen Devin L , Ma Weikang , Kass David A , Irving Thomas C , Day Sharlene
Published: October 2023

Significance:
While the disease mechanisms of truncating MyBP-C variants in HCM have been well defined, the mechanisms by which missense MyBP-C variants cause HCM are largely unknown. Recent studies show that unlike truncating variants that push myosin into an active high energy state due to their overall reduction in MyBP-C, missense MyBP-C properly incorporates into the sarcomere without affecting protein abundance. The consequences of missense MyBP-C on myosin have not been explored and may have implications for the treatment of HCM patients with missense MyBP-C variants.

Results:
We performed small angle X-ray diffraction (XrD) on human HCM tissue from patients with missense MyBP-C, and healthy control hearts. When healthy tissue was moved from relaxing pCa 8 to activating pCa 5 the I
1,1
/I
1,0
equatorial intensity ratio increased (p<0.001) indicating myosin is moving away from the backbone and adopting an active conformation. However, in missense hearts I
1,1
/I
1,0
is lower than healthy hearts at pCa 8 meaning more myosin is localized to the backbone. This is contrary to other HCM mutations which are proposed to push myosin to an active conformation under relaxing conditions. I
1,1
/I
1,0
does not increase at pCa 5 (p>0.05) indicating that at activating Ca myosin remains in the inactive conformation suggesting the thick filament Ca response is impaired in missense hearts.

Ongoing Experiments:
We are planning to perform XrD on more samples and Mant-ATP assays to determine if the structural inactivation of myosin in missense tissue also results in myosin remaining in the low energy super relaxed state (SRX). We expect the structural state to correlate with the biochemical state meaning missense hearts will demonstrate an equivalent percentage of SRX myosin compared to control hearts.

Conclusions:
Our finding that the transition of myosin to a structurally active conformation does not occur in human MyBP-C missense hearts distinguishes these variants from other HCM causing mutations such as truncating MyBP-C and missense MYH7 which have been shown to shift myosin to the active disordered relaxed state. If Mant-ATP assays show myosin is maintaining SRX along with an inactive conformation this suggests MyBP-C missense mutations act through a different mechanism.
Abstract P1048: Lattice-Boltzmann Method Achieves Feasible Hemodynamic Analysis In Carotid Atherosclerosis With Low Computational Cost
Authors: Park Seung Hyun , Cho Hyunwoo , Ahn Hae Nam , Park Yeongchae , Kim Kwang Won , Cho Brian , Ha Hojin , Ko Han Seo , Kim Doosang
Published: October 2023

Introduction:
Atherosclerosis is a common cardiovascular pathophysiology that can lead to plaque-related complications, including stroke and myocardial infarction. Non-invasive hemodynamic analysis can provide valuable insights into the plaque's rupture vulnerability and growth progression. Computational fluid dynamics is widely studied to understand hemodynamic pathophysiology further; however, traditional computational fluid dynamics methods often require daily-based analysis time to visualize hemodynamic parameters. This study investigated the feasibility of using the lattice Boltzmann method (LBM) for hemodynamic analysis of atherosclerosis lesions.

Methods:
From ten carotid artery stenosis patients, 20 carotid artery models were generated and analyzed using LBM. Hemodynamic parameters, including time-averaged wall shear stress (TAWSS), oscillatory shear index (OSI), and relative residence time (RRT), were compared between the stenotic lesion and contralateral control sides. The patient-specific boundary conditions were applied. Their cardiac cycle was subdivided into three periods: systolic acceleration, systolic deceleration, and diastolic, and analyzed to investigate their relationship with the hemodynamic parameters.

Results:
LBM effectively captured the hemodynamic parameters associated with carotid atherosclerosis with an hourly-based computational cost (2.765 [2.435-3.233] hours). The analysis visualized high TAWSS and low RRT distribution at stenotic areas during the systolic deceleration period and low TAWSS and high RRT distribution at the post-stenotic distal lesion during the diastolic period. These findings were reproducible among the ten patients.

Conclusion:
LBM-based hemodynamic analysis has the potential to serve as a non-invasive diagnostic tool that can help understand atherosclerosis pathophysiology with low computational cost.
Abstract P1049: The Impact Of Endurance Exercise On Longer-term Myocardial Fibrosis Development And Ventricular Arrhythmogenicity In Murine Coxsackievirus B3 Myocarditis
Authors: Favere Kasper , Van Hecke Manon , Eens Sander , Bosman Matthias , Delputte Peter L , De Sutter Johan , Fransen Erik , Roskams Tania , Guns Pieter-Jan , Heidbuchel Hein
Published: October 2023

Aims:
Diagnostic work-up of athletes often reveals nonischaemic myocardial fibrosis, which is associated with cardiac dysfunction and malignant arrhythmias. Its isolated finding on magnetic resonance imaging is commonly attributed to preceding viral myocarditis. Previous murine studies have shown a negative effect of exercise early in the course of viral myocarditis. In this study, we investigate, for the first time, the impact of endurance exercise on myocardial fibrosis development and arrhythmogenicity in a murine viral myocarditis model.

Methods & Results:
Eleven-week-old male C57BL/6J mice (n=72) were randomised to 8 weeks of treadmill running (EEX) or without exercise (SED). After 2 weeks, animals received a single injection with either coxsackievirus B3 to induce acute viral myocarditis (CVB) or vehicle as control (PBS). Exercise resulted in a milder clinical disease course, with less weight loss and better preserved running capacity. In addition, mortality was lower in exercising myocarditis mice (11 vs. 27%), however without statistical significance (P=0.23). Exercise during myocarditis increased the occurrence of interstitial fibrosis (limited or extensive distribution) at sacrifice (
i.e.
6 weeks after inoculation)(82.4% in CVB-EEX vs. 56.3% in CVB-SED; P=0.049). Additionally, exercise enhanced development of perivascular and/or interstitial fibrosis with extensive distribution (64.7% & 64.7% in CVB-EEX vs. 50% & 31.3% in CVB-SED; P=0.048). CVB-EEX animals demonstrated more myocardial scars on average, albeit not significantly (1.9 vs. 1.2; P=0.19), with similar scar distribution. Despite manifest fibrotic remodelling, collagen and fibrogenetic genes were not upregulated in the myocarditis groups at the time of sacrifice.
In vivo
right ventricular programmed electrical stimulation showed comparable ventricular arrhythmia inducibility in the myocarditis groups (P>0.20). The longest arrhythmias and highest cumulative arrhythmia burden occurred in CVB-EEX, however, without reaching statistical significance when compared to CVB-SED (P=0.49).

Conclusion:
Endurance exercise during viral myocarditis enhances perivascular and interstitial fibrosis development, which may promote ventricular arrhythmogenicity.
Abstract P1053: Circulating Mitochondrial DNA Level Is A Biomarker Of Acute Myocardial Infarction And Mediates Cardiac Ischemia-reperfusion Injury
Authors: Chu Yuxin , Hua Yutao , Brown Jamelle , Bell Celeste , He Jin , He Lihao , Chen Yunxi , Prabhu Sumanth D , Zhang Cheng , Ballinger Scott , Xie Min
Published: October 2023

Introduction:
Ischemia/reperfusion (I/R) injury occurs after reperfusion, causing significant myocardial damage. Circulating mitochondrial DNA (mtDNA) levels are elevated in acute myocardial infarction patients, and act as Damage Associated Molecular Patterns (mtDNA DAMP) that induce inflammation via immune processes, including the toll-like receptor 9 (TLR9) pathway. However, mtDNA DAMP levels have not been measured in ST-elevation myocardial infarction (STEMI) patients before and after percutaneous coronary intervention (PCI) and the role of mtDNA DAMPs in I/R injury remains unknown.

Hypothesis:
Pre- and post-PCI levels of mtDNA DAMPs serve as markers of STEMI related cardiac injury. Limiting mtDNA DAMP levels by increasing autophagy or blocking their downstream effects on TLR9 will decrease cardiac I/R injury.

Methods:
Troponin and mtDNA DAMP levels were measured pre- and 24 hours post- PCI in 55 STEMI patients (90%-100% blockage) and compared to 37 healthy controls. MtDNA DAMP levels in cell-free plasma were measured by real-time qPCR. Using a C57BL6J mouse model of I/R injury, Tat-Beclin (TB, an autophagy inducer), ODN2088 (a TLR9 inhibitor) or negative control were given at reperfusion and infarct size, cardiac function, and mtDNA DAMP levels were determined.

Results:
Pre- PCI mtDNA DAMP levels were increased ~200 folds (p<0.0001) in STEMI patients compared to healthy controls; post- PCI mtDNA DAMP levels were significantly decreased in the majority of patients. In contrast, troponin T levels increased, suggesting that mtDNA DAMP levels are early markers of STEMI. Interestingly, a greater decrease in mtDNA DAMP levels was associated with shorter symptom-to-PCI time. In mice, both TB induction of autophagy and TLR9 blockage by ODN2088 decreased infarct size by ~50% and preserved systolic function. As expected, TB induction of autophagy decreased mtDNA DAMP levels, while ODN2088 had no impact on mtDNA DAMP levels.

Conclusions:
Circulating mtDNA DAMP level is an early marker of STEMI and may predict the success of early PCI. Moreover, limiting the downstream effects of mtDNA DAMPs via induction of autophagy or blocking their action on TLR9 ameliorates cardiac I/R injury, and thus may provide a viable therapeutic avenue for STEMI patients.
Abstract P1055: Takotsubo Syndrome Model In Rats
Authors: Zulfaj Ermir , Nejat Amirali , Haamid Abdulhussain , Espinosa Aaron , Hussain Shafaat , Elmahdy Ahmed , Kakei Yalda , Redfors Bjorn , Omerovic Elmir
Published: October 2023

Background:
Takotsubo syndrome (TS) is an acute form of heart failure. The pathophysiology of the life-threatening syndrome is partially understood. A relevant animal model can aid in elucidating mechanisms behind TS. Current models of TS lack proper reproducibility, have high mortality, and fail to reflect the human condition adequately. This experimental study aimed to produce a human-like TS model.

Methods:
Rats were infused i.v. with a single dose of isoprenaline in varying doses (0.1-50 mg/kg) and infusion times (1-60 min). High-resolution speckle-tracking echo and 3D imaging was used to study heart function for up to 30 days.

Results:
Mechanical dyssynchrony preceded global contractile dysfunction and development of transient apical akinesia. Surviving rats showed normal behaviour, ECG and imaging at day 30. TS-like phenotype was achieved in 92.9% of survivors. Mortality at 30-day was 6.7%. The deceased animals developed complications like TS patients including heart failure, malignant arrhythmias, mural thrombus formation, cardiogenic shock. A dose-dependent relationship in incidence (p<0.0001), extent of akinesia (p<0.01), mortality (p<0.01), and cardiac function was seen with stress hormone. Systolic cardiac function at first echo was a good predictor for mortality (Odds Ratio 0.73; 95% CI 0.57-0.85), while the extent of akinesia strongly correlated with time until recovery (Rho = 0.86).

Conclusions:
This study provides a highly reproducible TS model with low mortality and excellent translational potential. High-resolution imaging and the natural course demonstrate the presence of the most critical characteristics of TS-like phenotype as in humans.
Abstract P1059: Decellularization Of Valved Porcine Xenografts For Use In Pediatric Right Ventricular Outflow Tract Replacement Surgery
Authors: Harris Amy G , Bartoli-Leonard Francesca , Iacobazzi Dominga , Carraba Michele , Biglino Giovanni , Caputo Massimo
Published: October 2023

Introduction:
The right ventricular outflow tract (RVOT) is pathologically affected in multiple congenital heart diseases, requiring surgical replacement within the first year of life. Shortage of small-sized homografts has driven xenograft use in pediatric cardiac surgery. However, these RVOT substitutes do not possess lifelong durability, eliciting high levels of inflammation and lacking growth potential. Xenograft limitations culminate in structural graft deterioration and rejection, resulting in invasive reoperations. Decellularization of donor tissue to eliminate cells and immunogenic antigens is a central method by which rejection risk can be diminished. Maintenance of the extracellular matrix (ECM) must be balanced with achievement of acellularity. Currently, no xenograft has proven optimal for pediatric RVOT surgical replacement; this work aims to produce a resilient and non-immunogenic decellularized porcine scaffold with translational capacity.

Methods/Results:
Research sought to optimize a combined chemical, enzymatic, and mechanical decellularization approach for RVOT conduits. The nine-day protocol utilizes a temperature-controlled bioreactor wherein EDTA, protease inhibitors, sodium dodecyl sulfate, and nucleases are circulated through the lumen of the submerged conduit. Maintenance of ECM structure, including elastin and collagen, in parallel with significant nuclei count reduction, removal of residual nucleic acids, and elimination of xenoantigens has been achieved. No significant difference was found between control and decellularized ventricular soluble collagen. All comparisons between samples processed directly after explant (fresh) and those decellularized following one freeze-thaw cycle (freeze-thawed) were not significantly different.

Conclusion:
Data indicate successful decellularization of valved conduits. Future work intends to seed mesenchymal stem cells onto the cell-free scaffold, improving biocompatibility and endowing the xenograft with growth potential. Decellularized RVOT xenografts provide a means to combat immunological mismatch between donor and patient, reducing the number of surgeries a child is exposed to and improving pediatric congenital surgery outcome.
Abstract P1060: Correlation Of Plasma Glutathione Peroxidase 4 Concentration With Coronary Heart Disease And The Severity Of Coronary Artery Stenosis
Authors: Song Dan , Yunus Rayaan , Mahmood Faraz , Bu Yifan , Li Jia , Matyal Robina , Xie Hebin , Leng Yiping , Deng Ping , Zhong Qiaoqing
Published: October 2023

Introduction:
Selenium-dependent glutathione peroxidase (GPX4) is an antioxidative, monomeric enzyme that can protect cells from membrane lipid oxidation-linked damage. Given the potential influence of GPX4 concentration on cardiovascular risk, we investigated how GPX4 levels change in patients with coronary heart disease (CHD), and whether GPX4 concentration correlates with the severity of coronary artery stenosis (CAS).

Methods:
Patients admitted at our institution between August 2022 and January 2023 for suspected CHD and completed coronary angiography were included. Patients were stratified according to their coronary angiography results: CHD group (positive) and control group (negative). The severity of CAS in patients with CHD was evaluated by dividing patients into low (<43 points) or high (≥43 points) Gensini score groups. Plasma GPX4 concentration was measured by enzyme-linked immunosorbent assay (ELISA).

Results:
Eighty-one patients were included based on our eligibility criteria, 41 patients in the CHD group and 40 patients in the control group. Patients with CHD were more commonly male and hypertensive, and presented with significantly greater levels of triglycerides and LDL-cholesterol at baseline. ELISA analysis indicates that patients with CHD have significantly lower plasma GPX4 concentrations as compared to patients in the control group. Furthermore, Spearman’s correlation analysis shows an inverse association between plasma GPX4 concentration and Gensini score in patients with CHD.

Conclusion:
Our results suggest that plasma GPX4 concentration was significantly lower in patients with CHD and is negatively correlated with the severity of CAS.
Abstract P1067: A Perfused
In Vitro
Model Of Embryonic Heart Tube: Leveraging 3D Bioprinting And Human Induced Pluripotent Stem Cell Technologies To Study Heart Development
Authors: Jin Linqi , Tomov Martin L , Chen Huang , Avazmohammadi Reza , Dasi Lakshmi , Bauser Heaton Holly , Serpooshan Vahid
Published: October 2023

Human heart development is an intricate dynamic process that remains barely understood, due to the limited access to the developing human heart
in utero
and the lack of effective modeling systems. Rapid advances in 3D bioprinting and stem cell technologies have enabled the fabrication of cardiovascular tissues with complex structures and microenvironment. Here we present a perfusable model of human embryonic heart tube manufactured using digital light processing (DLP) 3D bioprinting. The bioprinted model contains an endocardium, a cardiac jelly, and a myocardial layer resembling the structure of the human linear heart tube on day 22 (
Figure 1A-D
). Human iPSC-derived cardiomyocytes (hiPSC-CMs) and umbilical vein endothelial cells (HUVECs) were used to cellularize the constructs (
Figure 1F
). Full endothelialization of the heart tube lumen and expression of cardiac troponin T in the myocardial layer were confirmed via immunofluorescent staining (
Figure 1F
). Global tissue contraction in culture demonstrated the viability and cardiac function of the engineered model. Single-cell RNA sequencing further examined the gene expression profiles across different experimental groups. This study establishes the first perfusable model of human embryonic heart tube, which can serve as a robust platform to decipher the complex cellular processes underlying early heart development and the potential impact of microenvironmental factors on these events.

Figure 1: A-D:
Design of a developing human heart model on embryonic day 22.
E-H:
The workflow to bioprint (
E
), cellularize (
F
), and conduct dynamic culture (
G-H
) of the embryonic heart models using a perfusion bioreactor system.
Abstract P1076: Rrad Regulates Cardiomyocyte Cell Cycle Entry By Modulating Calcineurin Activity
Authors: Devilee Lynn , Miller Jessica , Ou Qinghui , Salama Abou Bakr M , andres douglas , Satin Jonathan , Mohamed Tamer , Hudson James E , Abouleisa Riham
Published: October 2023

Background:
Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. We and others have shown that reduced CM contractility can promote cell cycle induction; however, the underlying mechanisms are still unclear. Calcium (Ca
2+
), a key regulator of cardiac contractility, is also an important signal transducer that regulates key cellular processes. Ca
2+
enters CMs through the LTCC, which triggers Ca
2+
release from the sarcoplasmic reticulum to initiate CM contractility. The aim of this study is to understand the role of Ca
2+
signaling during CM cell cycle induction.

Methods and Results:
We performed a drug screening targeting proteins involved in CM calcium cycling in human iPS-derived cardiac organoids. Changes in Ki-67 expression were assessed as an S-G2/M phase marker. The top hit was Nifedipine, an inhibitor of LTCC. To inhibit the LTCC activity, we overexpressed Ras-Related Associated with Diabetes (RRAD), an endogenous regulator of LTCC activity. RRAD overexpression significantly reduced the intracellular calcium amplitude. RRAD overexpression promoted cell cycle induction, increased CM number, and upregulated cell cycle genes in primary neonatal, adult pig, human heart slices, and human iPS-derived cardiac organoids. Moreover, RRAD overexpression significantly augmented the CM response to other cell cycle stimulators such as Cyclin A2, a combination of CDK4 and cyclin D (2F), or a combination of CDK1, CDK4, cyclin D, and cyclin B (4F). Mechanistically, we found that RRAD overexpression reduced the activity of Ca
2+
dependent serine/threonine phosphatase (Calcineurin), as indicated by a significant reduction in calcineurin phosphatase activity
in vitro
and the expression levels of the calcineurin downstream effectors such as Rcan1 expression as well as translocation of NFAT and Hoxb13 to the CM nucleus.

Conclusion:
Disruption of Ca
2+
handling by RRAD overexpression promotes cell cycle induction in CMs through modulation of calcineurin activity. Further work
in vivo
is needed to test whether RRAD overexpression could improve cardiac function after ischemic injury.
Abstract P1082: The Role Of Shroom3 Protein In Cardiac Regeneration And Repair
Authors: Bakhshian Nik Amirala , Jamal Ayana , Link Brian , Omeara Caitlin
Published: October 2023

The cardiac injury response mainly depends on species and developmental stage. In a pro-regenerative context, including zebrafish and neonatal mice, following cardiac injury and death of cardiomyocytes (CMs), remaining CMs re-establish a functional cytoarchitecture, proliferate, and integrate into a re-muscularized heart. However, in a non-regenerative context, existing CMs do not readily proliferate, but instead, remodel their cytoarchitecture and undergo hypertrophy. Of note, CM cytoskeletal structure and intercalated disc (ICD) play a key role in regulating cell signaling and cardiac function. In zebrafish, Shroom3 (Shrm3) is a protein that mediates CM morphology, proliferation, and ICD integrity. Previous studies suggest that Shrm3 deletion results in increased CM proliferation in embryonic mice and zebrafish. Here, we investigate the function of Shrm3 on CM and its role in mediating a regenerative response to myocardial injury. To date, the role of Shrm3 in regulating the postnatal CM cell cycle has not been explicitly explored due to high lethality in global Shrm3 knockout mouse models. Thus, we developed a tamoxifen-inducible CM-specific Shrm3 deletion mouse model (Shrm3
fl/fl
;Myh6
MCM
) to study the Shrm3 role in both neonate and adult mice. At P7, Shrm3
fl/fl
;Myh6
MCM
uninjured mice showed increased EdU incorporation in CM nuclei compared to wildtype controls suggesting that as in zebrafish and embryonic mice, Shrm3 knockout increased CM cell cycle activity postnatally. We performed apical resection injury on P1 mice and at 21 days post-injury (dpi) Shrm3
fl/fl
;Myh6
MCM
CMs displayed compromised ICDs. Trichrome staining of 21 dpi hearts revealed minimal scar formation in Shrm3
fl/fl
;Myh6
MCM
mice, however, we observed aberrant regeneration of the ventricular apex as indicated by invagination of the cardiac apex and significantly reduced ejection fraction at 21 dpi. Thus, our data suggest that Shrm3 deletion in postnatal CMs remodels cell junctions and increases CM cell cycle activity. Our future studies will address the Shrm3 function(s) in cardiac regeneration and repair in injured adult mice to develop potential therapeutic strategies.
Abstract P1084: Cardiomyocyte-specific Overexpression Of Ccnd2 Via Modified Mrnafor Remuscularization In Hearts With Myocardial Infarction
Authors: Sun Jiacheng , Wang Lu , Matthews Rachel , Walcott Gregory , LU YU-AN , Wei Yuhua , Zhou Yang , Zangi Lior , Zhang Jianyi J
Published: October 2023

Introduction:
The adult mammalian heart has limited regenerative capacity due to postnatal cardiomyocyte (CM) cell cyclearrest. With the success of modified mRNA (modRNA) vaccine against SARS-CoV-2, modRNA technology offers efficient, transient, safe, nonimmunogenic, and controlled mRNA delivery without any risk of genomic integration. Using our previously reported cardiomyocyte specific modRNA translation system (CM SMRTs), we investigated the myocardial regenerative potential and therapeutic outcome of transient and specific cardiomyocyte overexpression of cyclin D2 (CCND2) in a pig model of myocardial infarction (MI).

Methods:
Modified mRNA was synthesized by in vitro transcription. AMI was induced by occlusion of the left anterior descending coronary artery for 60 minutes with reperfusion. Pigs were randomly assigned to vehicle group (1ml sucrose-citrate buffer, n=6), nGFP-CM SMRTs group (7.5mg CM-specific nuclear GFP modRNA, n=7)and CCND2-CM SMRTs group (7.5mg CM-specific CCND2 modRNA, n=8). CM cell cycle activity was measured 3 days after injection by immunostaining for proliferation marker Ki67, M-phase marker phospho-histone H3 (PH3) and cytokinetic marker aurora B kinase (AuB). Cardiac function was evaluated by echo and cardiac MRI (cMRI) on day 28; infarct size was determined via late gadolinium enhancement cMRI; arrhythmia incidence was assessed via implanted loop recorders.

Results:
The short-term in vivo study demonstrated a highly specific overexpression of targeted genes in CMs. CCND2-CM SMRTs significantly promoted CM cell cycle activation that was evidenced by significant increase of cell cycle markers compared with nGFP-CM SMRTs group: Ki67 (4.21±0.34% vs. 0.81±0.05%), PH3 (0.51±0.06% vs. 0.13±0.03%), and Symmetric AuB (0.035±0.007/mm
2
vs. 0.000±0.000/mm
2
), 3 days after AMI and modRNA treatment. The long-term follow-up of the animals also showed improved heart function (EF: 50.34±1.94% vs. 38.60±1.18%) and decreased scar size (8.80±1.07% vs. 22.77±2.77%). The frequency of arrhythmic events in animals between all groups were similar.

Conclusion:
The results in large animal model of AMI demonstrate remuscularization and therapeutic potential of cardiomyocyte-specific overexpression of CCND2 via modRNA.
Abstract P1085: Generating Injectable Vascularized Cardiomyocytes Derived From Human Induced Pluripotent Stem Cells
Authors: Aoyama Junya , Sokol Morgan , Zhu Anthony , Cao Fang , Gil Eun S , Aleksi Elton , Garbern Jessica , Elwell Hannah , Barrera Irving , Lee Richard T
Published: October 2023

Introduction:
Regenerated cardiomyocytes (CMs) derived from human-induced pluripotent stem cells (hiPS-CMs) have potential for improving deficits in cardiac function due to loss of cardiomyocytes. However, hiPS-CMs are immature relative to adult cardiomyocytes. Here, we demonstrate that injecting CMs with self-assembling peptide (SAP) nanofibers can promote vascularization, organization of sarcomeres, and maturation of the CMs.

Methods:
We first cultured hiPS-CMs on different SAPs in vitro and analyzed gene expression related to CM maturation. We then implanted hiPS-CMs with the SAP into uninjured rat left ventricular (LV) wall and evaluated sarcomere organization and vascularization up to three months. In addition to uninjured myocardium, we also evaluated the effects of SAPs in post-ischemic rat myocardium. Furthermore, we analyzed the benefit of SAPs on CM maturation of injected hiPS-CMs within the rat LV wall by the Slide-seq spatial single-cell genomics technique.

Results:
Among SAPs tested, the RADA16 peptide promoted mRNA expression related to CM maturation, including expression of myofibril isoform subunit
MYH7/6
, gap junction
GJA1
, and potassium ion channel
KCNJ2
. Moreover, the expression of
HCN4
, a gene expressed in pacemaker cells, decreased on RADA16. The hiPS-CMs injected with RADA16 had improved organization of sarcomeres and more vascularization up to 3 months. More vascularization by RADA16 with hiPS-CMs was also confirmed in the ischemic LV wall. Slide-seq revealed that the maturation of hiPS-CMs occurred earlier with co-injection with the RADA16.

Conclusion:
The SAP RADA16 promoted improved maturation of CMs in vitro. Furthermore, this SAP promoted the organization of sarcomeres, maturation of myofibrils, and vascularization in vivo, which could benefit implanted hiPS-CMs in improving host cardiac function.
Abstract P1109: DNA Damage-free iPS-Cardiomyocytes Reduces Cardiac Fibrosis
Authors: Miller Jessica , Krishnamurthy Prasanna , Sethu Palaniappan , Kannappan Ramaswamy
Published: October 2023

With the increasing potential of employing cardiomyocytes (CMs) derived from human induced pluripotent stem cells (iPSC) in myocardial repair, there is a demand for a method of reducing the induction of fibrosis at the transplantation site. We hypothesized that transplantation of anti-inflammatory DNA damage-free CMs (DdF-CM) could reduce fibrosis formation at the implantation site. The transcription factor, p53, is inflammation-dependent: influencing apoptosis in inflammatory or damaged cells and promoting proliferation in normal cells. Previously, we demonstrated that the p53 activator nutlin-3a could prime hiPSC culture to contain only DdF-iPSCs by inducing apoptosis in DNA-damaged inflammatory cells and successfully differentiated them into DdF-CMs. Further investigation into these cells revealed that DdF-CMs secrete exosomes containing miR-26a. These miR-26a exosomes are up-taken by host fibroblasts, reducing collagen I secretion around the graft site and overall scar size formation post-MI. Additionally, DdF-CMs transplanted mice demonstrated improved cardiac function and cell engraftment after 4 weeks post-MI. This study illustrates that transplanting DdF-CMs into infarcted hearts improves cardiac function and reduces fibrosis formation at the implantation site.
Abstract P1143: Med12 “Mediates” Transcriptional Regulation In The Heart
Authors: Kolonay Dominic , Baskin Kedryn K
Published: October 2023

The Mediator complex serves as a bridge to link transcriptional machinery and transcription factors (TFs) to control transcription, but the molecular mechanisms of transcriptional regulation by Mediator are not well-understood. Mediator is a large multiprotein complex organized as submodules, known as the head, body, tail, and kinase submodule. The CDK8 kinase submodule is known to regulate Mediator - RNA Polymerase II interaction. MED12 is an essential Mediator component, and within the kinase submodule, is required for CDK8 kinase activity. MED12 protein levels are increased in human heart failure biopsies and in the mouse heart after transverse aortic constriction (TAC)-induced heart failure. To test the hypothesis that increased levels of MED12 contributes to development of heart failure we generated cardiac-specific
Med12
transgenic mice (cTg) by driving increased MED12 expression in cardiomyocytes using the αMHC promoter.
Med12
overexpression leads to decreased cardiac function, measured as fractional shortening by serial echocardiography. Decreased cardiac function was accompanied by increased cardiac chamber size and dilation, without cardiac fibrosis. RNA sequencing of cardiomyocytes from cTg and control mice revealed that increased levels of MED12 leads to dysregulated calcium handling gene expression. MED12 is a transcriptional regulator, but does not directly bind DNA. To determine how MED12 regulates gene expression we screened for TFs that interact with MED12. We performed immunoprecipitation of MED12 and by mass spectrometry and determined that MED12 interacts with the TFs MEF2, CREB, and SRF. In follow-up experiments we demonstrated that MED12 interacts with the transcription factor (TF) MEF2 to regulate calcium handling genes. Current studies are focused on determining how MED12 binding coordinates gene expression through multiple TFs in cardiomyocytes. Collectively, our data demonstrate that increased MED12 expression leads to the development of heart failure, in part by dysregulation of gene expression. Therefore, precise regulation of MED12 levels is important to maintain normal cardiac function.
Abstract P1148: Glucocorticoid Receptor - Klf9 Axis Regulates Cardiomyocyte Metabolic Homeostasis
Authors: Thakkar Chandni , Alikunju Saleena , Rizvi Wajiha , Abbas Ali , Yang Zhi , Sayed Danish H
Published: October 2023

Glucocorticoids (GCs) through activation of the Glucocorticoid receptor (GR) plays an essential role in cellular homeostasis during physiological variations and in response to stress. GC-GR signaling is involved in regulating several cellular processes including metabolism, circadian rhythm and inflammation for diurnal adaptations. Our genomic GR binding (ChIP) and transcriptome (RNAseq) data from Dexamethasone (Dex) treatment in cardiomyocytes show an early (1hr) differential regulation of mostly transcription factors, followed by sequential change in downstream signaling pathways (6-12hr). Here, we examine the role of an early direct target of GR in cardiomyocytes, Krüppel-like factor 9 (Klf9) in metabolic homeostasis. Our Klf9-ChIPseq identified 4100 genes with change in promoter Klf9 binding in response to Dex. Functional annotation of these genes lists metabolic pathway on the top of KEGG pathway, along with genes regulating transcription and survival. Interestingly, integration of GR and Klf9 data show overlapping targets (1181), suggesting that Klf9 could be serving as feedback regulator for these genes. Further, our transcriptome analysis of Dex treated cardiomyocytes with knockdown of Klf9 reveal differential regulation of 1777 genes (Dex+siKLf9 vs Dex+siLUC), of which a reversal in expression is seen in 1640 (92%) genes with siKlf9
vs.
Dex. Conversely, only 137 (8%) genes show further dysregulation in expression with siKLf9
as
Dex. Gene ontology of these 1640 genes show metabolic pathway on the top, including genes involved in glycolysis and oxidative phosphorylation. Conversely, mostly immuno-regulatory genes are among those 137 genes. Expectedly, knockdown of Klf9 in cardiomyocytes inhibits Dex induced increase in glycolysis and glycolytic capacity by 24%, and spare respiratory capacity by 50%, as measured by ECAR and OCR with glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GC mediated GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular metabolic homeostasis. With hypertrophic stress a decrease in cardiac GR and dysregulated Klf9 signaling might result in metabolic crisis, contributing to mitochondrial and cardiac dysfunction
Abstract P1152: Molecular Regulation Of Ferroptosis In The Type 1 Diabetic Heart
Authors: Gawargi Flobater I
Published: October 2023

Background/Hypothesis:
The molecular mechanisms underpinning ferroptosis, a newly emerged mechanism of myocardial cell death, remains unclear in the T1DM heart. Although the targeted deletion of matrix metalloproteinase-9 (MMP9) is cardioprotective, the role of MMP9 in T1DM-induced cardiac ferroptosis is unknown. We hypothesize that the lack of MMP9 mitigates ferroptosis in the T1DM heart.

Methods/Results:
We randomly selected 15-week-old male

Ins2
+/-

Akita (T1DM) and age and gender-matched
Ins2
+/+
littermate WT mice to examine T1DM-induced cardiac ferroptosis. We used our novel Akita/MMP9KO (DKO) mice to investigate the specific effect of MMP9 deficiency on T1DM-induced cardiac ferroptosis. Following the
guidelines on myocardial cell death
, we evaluated ferroptosis in the heart (LV) of WT, Akita, DKO, and MMP9KO mice. The comparison of protein levels in (i) Akita vs. WT and (ii) Akita vs. DKO (n=3-6) revealed that the lack of MMP9 prevents T1DM-induced upregulation of iron import [Transferrin Receptor (TFR): (i) +2.44-fold, (ii) -1.78-fold], downregulation of iron export [Ferroportin-1: (i) -3.20-fold, (ii) +2.20-fold], amplification of lipid peroxidation via a decrease in Glutathione Peroxidase-4 (GPX4) [(i) -1.95-fold, (ii) +2.01-fold], antioxidants [ {Superoxide dismutase-1 (SOD1): (i) -1.1-fold, (ii) +1.00-fold} and SOD2 {(i) -1.58-fold, (ii) +1.01-fold}], and an increase in Lysophosphatidylcholine Acyltransferase-3 (LPCAT3) [(i) +3.14-fold, (ii) -2.09-fold] in the heart.

Conclusion:
We reveal the mechanisms by which MMP9 deficiency mitigates ferroptosis in the T1DM heart (Figure 1). This is the first report of MMP9 role in cardiac ferroptosis.
Abstract P1156: Potential Role Of Ferroptosis In Duchenne Muscular Dystrophy-associated Cardiomyopathy
Authors: Fefelova Nadezhda , Mareedu Satvik , Pamarthi Sri Harika , Babu Gopal J , Gwathmey Judith K , Xie Lai-Hua
Published: October 2023

Background and Aims:
Duchenne muscular dystrophy (DMD) is the most common and fatal form of muscular dystrophy. DMD-associated cardiomyopathy (DMD-CM) is a major cause of morbidity and mortality in patients with DMD. Ferroptosis, a new type of regulated cell death characterized by iron-dependence and the accumulation of cell membrane lipid peroxidation, has been manifested in various cardiovascular disease conditions. However, ferroptosis has not been studied in DMD-CM. Here, we aim to determine whether ferroptosis play a potential role in DMD-CM.

Methods and Results:
We used a dystrophin/utrophin double knockout (mdx:utr
-/-
) mouse model that displays clinical signs of DMD and DMD-CM. We performed RNA isolation from the ventricles of 3-month-old wild type (WT) and mdx:utr
-/-
mice (n=3) and performed RNA-Seq analysis. We found alterations of ferroptosis-related pathways such as GTP cyclohydrolase 1, transferrin receptor, glutamine transporter SLC38A1, heme oxygenase-1 (HMOX-1), and arachidonate 5-lipoxygenase (ALOX5) pathways in mdx:utr
-/-
hearts compared to WT hearts. Western blots confirmed the upregulation of ALOX5 and downregulation of HMOX-1, ferroptosis suppressor protein 1, and NADPH oxidase 4 in mdx:utr
-/-
hearts, while there was no change in glutathione peroxidase 4 (GPX4) levels. We also observed an increased level of lipid peroxidation in mdx:utr
-/-
hearts compared to WT as estimated by the level of malondialdehyde. Live/dead viability assays demonstrated a higher cell death rate in cardiomyocytes from mdx:utr
-/-
mice (53.7±7.2%, n=24) compared to those from WT mice (11.7±1.1%, n=17, p< 0.01) under control conditions. The observed cell death in mdx:utr
-/-
cardiomyocytes was efficiently prevented by the selective ferroptosis inhibitor ferrostatin-1 (10 μM) (7.4±1.5%, n=9, p<0.01), but not by the apoptosis inhibitor emricasan (20 μM) (56.0±1.6%, n=3, NS). Similarly, higher death rates were also observed in mdx:utr
-/-
than in WT cardiomyocytes when they were treated with various ferroptosis inducers such as iron overload and the GPX4 inhibitor RSL3.

Conclusion:
We have deciphered the link between ferroptosis and DMD-CM for the first time. Our data suggest novel targets for the treatment of DMD-CM by inhibiting ferroptosis.