bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP254: Decellularized Extracellular Matrix Hydrogel Lowers Fibrosis And Fibroblast Differentiation In Post-injury Mice Hearts Through Capg
Authors: Wang Xinming , Senyo Samuel
Published: November 2021

Hypothesis and objective:
We hypothesize that transplantation of decellularized cardiac extracellular matrix (dECM) lowers fibrosis and fibroblast differentiation. In this study we investigated collagen deposition and fibroblast differentiation in post-MI hearts and heart explants of various stiffness after dECM hydrogel treatments. The objectives are 1) determining if dECM derived from fetal and adult porcine hearts reduces fibrosis in injured hearts; and 2) identifying specific signaling pathways that regulate fibroblasts differentiation induced by extracellular proteins.

Methods:
Porcine dECM was injected immediately after ligating coronary artery in P1 mice. Histology was conducted on day 7 post-myocardial infarction (MI). A mice ventricle explant model was used to investigate the molecular mechanisms.

Results:
We observed that fetal dECM treatment lowered fibrosis and fibroblast differentiation in post-MI hearts (Fig.1). Fibroblast differentiation as indicated by α-smooth muscle actin expression in vimentin or platelet derived growth factor receptor α positive cells showed an inhibitory effect of fetal dECM on fibroblast differentiation. Using a heart explant model of modulated microenvironment stiffness, we demonstrated that increasing tissue stiffness stimulates fibroblast differentiation and collagen deposition. Fetal dECM treatment, however, inhibited fibroblast differentiation induced by increasing microenvironment stiffness. Transcriptome analysis revealed that two cytoskeleton-related genes, macrophage capping protein (CAPG) and leupaxin (LPXN), are modulated by dECM treatments. Using cytoskeleton polymerization modulators and siRNA, we demonstrated that fetal dECM lowers fibroblast differentiation through CAPG.
Abstract MP255: Intercellular Model Predicts Macrophage Signaling As A Driver Of Cardiac Fibroblast Response Post Myocardial Infarction
Authors: Chowkwale Mukti , Saucerman Jeffrey J
Published: November 2021

Introduction:
Post-myocardial infarction (MI), cardiac fibroblasts and macrophages work together to regulate tissue homeostasis and infarct repair. Macrophage-fibroblast interactions in healthy tissue are stable and resistant to perturbations. However, this robustness post-MI has not been assessed. This study designs and implements an intercellular communication model of macrophage-fibroblast crosstalk to determine drivers of infarct repair.

Methods:
An ordinary differential equation model of post-MI cellular dynamics was developed (
Figure 1A
). Model inputs are time courses of cardiomyocytes, neutrophils, and monocytes. These cells, along with simulated macrophages and fibroblasts, secrete chemokines and cytokines which directed cell proliferation, removal, and chemical secretion. The outputs are macrophage and fibroblast densities, secreted factor dynamics, and produced collagen. Model validation was done using published data in post-MI mice. A sensitivity analysis was conducted by knocking down individual parameters to identify key drivers of fibroblast collagen production.

Results:
The simulated trends matched the validation time courses. Of the 28 validation relationships, 12 were input-output, 7 were knockouts, and 9 were inhibitor relationships. The validation passed at 78.5%; the 6 failed validations were due to the independent nature of the input curves. Sensitivity analysis (
Figure 1B
) identified macrophage differentiation rate, removal rate, and transforming growth factor-beta (TGFB) secretion rate as pro-fibrotic. Prolonged exposure to TGFB and granulocyte-macrophage colony stimulating factor was anti-fibrotic. Several clustered parameters differentially regulated macrophages and collagen production.

Conclusions:
The multicellular model identified macrophage density as a pro-fibrotic driver in the healing infarct. Differential regulation of macrophages and collagen production was predicted by the model.
Abstract MP256: CCCTC-binding Factor Site Mutation In
Sirt1
Promoter Reduces Systolic Cardiac Function In Mice
Authors: Wagner Tobias , Micheletti Rudi , Taylor Havilah , Suh Yousin , Rosenfeld Michael G , Zemljic-Harpf Alice E
Published: November 2021

Regulation of the geroprotective histone-deacetylase SIRT1 is an important physiological strategy to protect against cardiovascular disease. SIRT1 activity regulates the oxidative stress response, DNA damage pathways, cell death, and cardioprotective SIRT1 levels decreases with age. Based on this and GWAS studies implicating misregulation linked to myocardial infarction risk, we hypothesized that disrupting proximal promoter regulation of
SIRT1
causes cardiac stress. Focusing on Single Nucleotide Polymorphisms (SNPs) in the
SIRT1
promoter, we used reporter gene assays to test if the transcriptional regulation of
SIRT1
after oxidative stress is altered by the SNPs. Identifying a candidate A/G polymorphism at the -92 position in the promoter of
SIRT1
, we show that the presence of the minority allele causes a disrupted epigenetic activation of
SIRT1
. In model cell systems, binding of CCCTC-binding factor (CTCF) in the promoter of
SIRT1
is reduced after oxidative stress in cells carrying the minority allele, correlating with a lack of activation of stress-protective
SIRT1
expression in iPSC-derived cardiomyocytes. To investigate the physiological consequences of this mutation, we used Crispr/Cas9 to generate global
Sirt1
promoter CTCF site mutant (Sirt1-mut) mice. Echocardiography in 7-month-old male mice shows reduced FS% (control 31.1±1.5%, Sirt1-mut 20.9±0.8%, mean±sem, p<0.0001, n=10-12 each), as well as increased LVIDs and decreased LVPWd wall dimensions in Sirt1-mut mice. RNA-seq on left ventricular cardiac tissue of 8-month-old control and Sirt1-mut mice identified 130 genes that are differentially regulated. Gene Ontology term enrichment analysis finds the differentially expressed genes are in pathways linked to regulation in response to reactive oxygen species and oxidative stress. In conclusion, we show that a mutation in the
Sirt1
promoter critical for oxidative stress responses leads to systolic cardiac dysfunction. The transcriptional regulation of cardioprotective SIRT1 is an important and hitherto overlooked factor in the pathophysiology of heart failure.
Abstract MP259: The Role Of Sertad4 In Pathological Cardiac Remodeling
Authors: Stratton Matthew , Francois Ashley , Bermeo-Blanco Oscar , Canella Alessandro , Marcho Lynn , McGrail Erin , Nishat Shamama , Whitson Bryan , Janssen Paul M , Accornero Federica , Gumina Richard J
Published: November 2021

Over 6 million Americans suffer from heart failure (HF) while the 5-year mortality rate following first admission for HF is over 40%. Cardiac fibrosis is a clinical hallmark of HF, regardless of the initiating pathology and is thought to contribute to disease progression. Using an epigenomics discovery approach, we uncovered a nuclear protein, Sertad4, as a potential anti-fibrotic target. Our data indicate that Sertad4 is a positive regulator of fibroblast activation. Specifically, cultured cardiac fibroblast experiments demonstrate that Sertad4 targeting with shRNAs blocks fibroblast proliferation and causes cells to arrest in the G2/M phase of the cell cycle. Also, shRNA targeting of Sertad4 dramatically blocked activation of myofibroblast differentiation genes (αSMA/POSTN/COL1A1). Mechanistically, these effects appear to be mediated by Sertad4 regulation of SMAD2 protein stability in the presence of TGF-β1 stimulation as demonstrated by proteasome inhibition experiments. RNA-seq analysis indicate that Sertad4 also regulates the expression of genes involved in ubiquitination and proteasome degradation. Next, we sought to determine the effect of global Sertad4 knockout on post-myocardial infarct (MI) remodeling and cardiac function in mice. After 4 weeks of permanent LAD ligation, echocardiography was performed to measure systolic function. Relative to wild-type (WT) controls, the Sertad4 KO mice showed preserved systolic function as evident by improved ejection fraction (WT 14.4 +/- 3.6 vs. KO 33.9+/-5.9, p=0.035) and fractional shortening (WT 6.5 +/- 1.7 vs. KO 16.4 +/- 3.4, p=0.046). β-gal staining in the Sertad4/LacZ reporter mouse subjected to MI showed robust Sertad4/LacZ expression in the ischemic scar and boarder-zone with almost no expression in control hearts. This data supports the notion that Sertad4 has a key role in cardiac remodeling in response to ischemic injury.
Abstract P303: Development Of
Casper/myl7/annexin-5
Transparent Transgenic In-vivo Zebrafish Model To Study The Cardiomyocyte Function
Authors: Rajpurohit Surendra K , Gopal Aaron , Mon May Y , Patel Nikhil , Arora Vishal
Published: November 2021

The Zebrafish provided an excellent platform to study the genetic and molecular approach ofcardiac research. Zebrafish heart cells similar to human heart cells at the molecular level anddetermine gene functions that control cardiac function and dysfunction. In zebrafish heart, myl7is myosin 7 gene and identified as a regulatory gene orthologs to human MYL7. In the heart,Annexin5 activities contribute to cardiomyocyte dedifferentiation, proliferation and epicardial injuryresponses which leads to cardiac cell death by apoptosis and narcosis pathways. We aredeveloping annexin-5 activity in the cardiovascular function under normal and in metabolicaberration by generating homozygous Casper/ myl7:RFP; annexin-5:YFP transgenic zebrafish.By developing
Casper/myl7/Annexin-5
transparent transgenic zebrafish model, we establish time-lapse in-vivo confocal microscopy to study of cellular phenotype/pathologies of thecardiomyocytes over time in newly developed strain to quantify changes in cardiomyocytemorphology and function overtime, comparing control and cardiac injury and cardio-oncologymodels. Transgenic zebrafish has normal type skin pigmentation background. In zebrafish,tracking of transgenic reporter activity in in-vivo is only possible in transparent stage. To maintaintransparency throughout the life, these strains crossbred with the skin transparent mutant Casper.Casper contributes to the study by integrating a transparent characteristic in adult zebrafish thatallows for simpler transparent visualization and observation. We develop casper transgenicprogenies through cross breeding with the transgenic strain of
myl7:RFP;annexin-5:YFP
.Confocal and fluorescent microscopy used to get accurate, precise imaging and to determinefluorescent protein being activated. 1.1: Generation of homozygous
casper
/
myl7:RFP;annexin-5:YFP
zebrafish (Generation F01-F05). 1.2: Screening and sorting the transgenic progeny andIn vivo imaging to validate cardiac morphology through in-vivo confocal imaging. Generation ofhomozygous
casper
/
myl7:RFP;annexin-5:YFP
zebrafish: Casper-Annexin5 homozygous stain:Cross breed casper and myl7/Annexin5 fish; F01: Generate the eggs from breeder and grow theembryo to attenuate larvae to screen for transgenic expression. F01 generation, larvae showtransgenic expression (47%). F02: transgenic expression larvae (39%). F02 heterozygous shownormal skin pattern; F03, larval show transgenic expression (43%). F04, transgenic larvae(90%).F04; 100% fishes are phenotypically casper; F05: heterozygous transgenic progeny togrow and continue to generate until achieve 100% homozygous casper-myl7-Annexin5 strain.These novel results provide in-vivo whole organism-based platform to design high throughputscreening and establish new horizon for drug discovery in the Cardiac Disease and Cardio-oncology.
Abstract P305: Patient-specific Ipsc-derived Cardiomyocytes Reveal Abnormal Regulation Of
Fgf16
In A Familial Atrial Septal Defect
Authors: Ye Lingqun , Yu You , Zhao Zhen-Ao , Zhao Dandan Zhao , Ni Xuan , Wang Yong , Fang Xing , Yu Miao , Wang Yongming , Tang Junming , Chen Ying , Shen Zhenya , Lei Wei , Hu Shijun
Published: November 2021

Aims:
Congenital heart disease (CHD) frequently occurs in newborns due to abnormal formation of the heart or major blood vessels. Mutations in the
GATA4
gene, which encodes GATA binding protein 4, are responsible for atrial septal defect (ASD), a common CHD. This study aims to gain insights into the molecular mechanisms of CHD using human induced pluripotent stem cells (iPSCs) from a family cohort with ASD.

Methods and Results:
Patient-specific iPSCs possess the same genetic information as the donor and can differentiate into various cell types from all three germ layers
in vitro
, thus presenting a promising approach for disease modeling and molecular mechanism research. Here, we generated a patient-specific iPSC line (iPSC-G4
T280M
) from a family cohort carrying a hereditary ASD mutation in
GATA4
gene (T280M), as well as a human embryonic stem cell line (ESC-G4
T280M
) carrying the isogenic T280M mutation using the CRISPR/Cas9 genome editing method. The GATA4-mutant iPSCs and ESCs were then differentiated into cardiomyocytes (CMs) to model GATA4 mutation-associated ASD. We observed an obvious defect in cell proliferation in cardiomyocytes derived from both GATA4
T280M
-mutant iPSCs (iPSC-G4
T280M
-CMs) and ESCs (ESC-G4
T280M
-CMs), while the impaired proliferation ability of iPSC-G4
T280M
-CMs could be restored by gene correction. Integrated analysis of RNA-Seq and ChIP-Seq data indicated that
FGF16
is a direct target of wild-type GATA4. However, the T280M mutation obstructed GATA4 occupancy at the
FGF16
promoter region, leading to impaired activation of
FGF16
transcription. Overexpression of
FGF16
in GATA4-mutant cardiomyocytes rescued the cell proliferation defect. The direct relationship between GATA4
T280M
and ASD was demonstrated in a human iPSC model for the first time.

Conclusions:
In summary, our study revealed the molecular mechanism of the GATA4
T280M
mutation in ASD. Understanding the roles of the GATA4-FGF16 axis in iPSC-CMs will shed light on heart development and provide novel insights for the treatment of ASD and other CHD disorders.
Abstract P307: An Exquisite Cardiac Performance And Protection Package Emerges In Mouse With Cardiac-specific Overexpression Of Adenylyl Cyclase Type 8
Authors: Qu Jia-Hua , Tarasov Kirill , Chakir Khalid , Lakatta Edward G
Published: November 2021

Young adult (three-month old) mice with cardiac-specific overexpression of AC type 8 (TGAC8) have a markedly elevated heart rate and markedly enhanced ejection fraction around the clock, mimicking cardiac responses of sympathetic autonomic input during acute exercise on a chronic base, but the TGAC8 mice do not exhibit heart failure or increased mortality up to about a year. Using this incessant young TGAC8 mouse as an ideal model to elucidate a high grade cardiac “Performance and Protection Package” (PPP) in response to chronic cardiac stress, we hypothesized that the TGAC8 heart adjusts to the chronical stress by reprogramming itself at multiple omics scales to code the exquisite cardiac PPP. To this end, we compared three-month old TGAC8 mice and their wildtype (WT) littermates using multiple omics analyses, from transcriptome to proteome, to phosphoproteome. Compared to WT, the phosphorylation level of most proteins was increased in the TGAC8 mice, including transcription factors (TF), kinases and phosphatases that are important in regulating transcription and phosphorylation. Among the 191 TFs identified in phosphoproteome, 91 were increased significantly, in line with the general upregulation in transcriptome. Many important stress response signaling pathways were enriched from phosphoproteome. Wherein, three protein quality control pathways, PI3K/AKT signaling, ERK/MAPK signaling and ubiquitination, were consistently enriched across the three omics scales. Most components in PI3K/AKT signaling pathway were upregulated, especially at protein and phosphorylation levels. Consistently, PI3K/AKT substrate phosphopeptides were increased, and downstream effects and functions of PI3K/AKT signaling were activated, including energy metabolism, protein synthesis, cell growth, cardiovascular functions, and NF-kB mediated functions. In summary, profiling the transcriptome, proteome and phosphoproteome of the TGAC8 heart unraveled the mechanism that controls its PPP. The cardiac overexpression of AC8 activates the AC8-cAMP-PKA axis to increase phosphorylation; widespread phosphorylation of the TFs promotes the transcription of numerous additional molecules that also regulate phosphorylation to reprogram the heart. Thereby, the TGAC8 mouse upregulates many stress response signaling pathways to activate the exquisite cardiac PPP.
Abstract P315: Shortening The Thick Filament By Partial Deletion Of Titin’S C-zone Alters Cardiac Function By Reducing The Operating Sarcomere Length Range Of The Heart.
Authors: Methawasin Mei , Farman Gerrie P , Granzier-Nakajima Shawtarohgn , Strom Joshua G , Smith John E , Granzier Henk L
Published: November 2021

Titin’s C-zone is the inextensible part of titin that binds along the thick filament at its cMyBP-C -containing region. Previously it was shown that deletion of titin’s super-repeats C1 and C2 (

Ttn
ΔC1-2

mouse model) results in shorter thick filaments and contractile dysfunction, but LV chamber stiffness is normal. Here we studied the contraction-relaxation kinetics from the time-varying elastance of the left ventricle (LV) and from cellular work loops of intact loaded cardiac myocytes. Ca
2+
transients were also measured as well as crossbridge cycling kinetics and Ca
2+
sensitivity of force. It was found that intact cardiomyocytes of

Ttn
ΔC1-2

mice exhibit systolic dysfunction and impaired relaxation. The time-varying elastance of the LV chamber showed that the kinetics of LV activation are normal but that relaxation is slower in

Ttn
ΔC1-2

mice. The slowed relaxation was, in part, attributable to an increased myofilament Ca
2+
sensitivity and slower early Ca
2+
reuptake. Dynamic stiffness at the myofilament level showed that cross-bridge kinetics are normal, but that the number of force-generating cross-bridges is reduced. In vivo sarcomere length (SL) measurements in the mid-wall region of the LV revealed that the operating SL range is shifted in

Ttn
ΔC1-2

mice towards shorter lengths. This normalizes the apparent cell and LV chamber stiffness but reduces the number of force generating cross-bridges due to suboptimal thin and thick filament overlap. Thus the contractile dysfunction in

Ttn
ΔC1-2

mice is not only due to shorter thick filaments but also to a reduced operating sarcomere length range. Overall these results reveal that for normal cardiac function, thick filament length regulation by titin’s C-zone is critical.
Abstract P316: Role Of Scleraxis In Cardiac Valve Development And Disease
Authors: Kodigepalli Madhav Karthik , Lincoln Joy
Published: November 2021

Valvular heart disease is one of the major causes of cardiac-related deaths in the US and effective treatment is currently limited to surgical repair or replacement. Mature heart valves are composed of highly organized and stratified layers of extracellular matrix (ECM) regulated by valve interstitial cells (VICs). This stratification that is initiated during embryogenesis and completed during valve maturation after birth, needs to be maintained throughout life for normal valve function. Myxomatous degeneration is a common disease histologically characterized by imbalance in ECM composition and organization resulting in valve biomechanical failure. Yet, key regulators of ECM homeostasis are not well characterized. Scleraxis (Scx) is a bHLH transcription factor that we previously showed to be critical for heart valve development and its loss of function leads to defective VIC maturation and aberrant ECM organization. However, due to lack of efficient tools, the mechanistic function of Scx in valve development and disease
in vivo
remains to be understood. Herein, we performed temporal analysis of
Scx
transcript levels during development of murine homeostatic valves and identified that Scx is predominantly expressed in the VICs between E13.5 and P14. We also assessed Scx levels and ECM abnormalities in valve tissues derived from patients with cardiac valve diseases and identified several distinct populations of VICs with high Scx levels that partially colocalized with elevated collagen fragmentation and proteoglycan (PG) deposition in the neighboring ECM. Scx levels were also elevated in valves of

Fbn1
C1039G/+

and osteogenesis imperfecta murine (
OIM
) mice that develop myxomatous valve abnormalities signifying a potential role in pathogenesis. To further study the function of Scx in valve development and disease
in vivo
, we have generated a conditional Scx-transgenic model (Scx-TG) that will allow for overexpression in targeted cell lineages upon
Cre
recombination. Additionally, we will employ Scx-Cre mouse model expressing Scx-promoter driven
Cre
-recombinase to perform Scx-expressing cell lineage analyses and high-throughput sequencing analyses to identify direct gene targets and protein-interaction partners of Scx to better understand its mechanistic role in regulating valve ECM homeostasis. Together these
in vivo
approaches will provide novel insights into the function of Scx in heart development and disease.
Abstract P317: Cardiac Fibroblast GSK3α Promotes Myocardial Fibrotic Remodeling Through GSK3α-ERK-IL11 Signaling Circuit
Authors: Umbarkar Prachi , Tousif Sultan , Singh Anand P , Anderson Joshua C , zhang qinkun , Lal Hind
Published: November 2021

Background:
Myocardial fibrosis contributes significantly to heart failure (HF). Fibroblasts are among the predominant cell type in the heart and are primary drivers of fibrosis. To identify the kinases involved in fibrosis, we analyzed the kinome of mouse cardiac fibroblasts (CF) isolated from normal and failing hearts. This unbiased screening revealed the critical role of the GSK-3 family-centric pathways in fibrosis. Previously we have shown that among two isoforms of GSK3, CF-GSK3β acts as a negative regulator of fibrosis in the injured heart. However, the role of CF-GSK3α in the pathogenesis of cardiac diseases is completely unknown.

Methods and Results:
To define the role of CF-GSK3α in HF, we employed two novel fibroblast-specific KO mouse models. Specifically, GSK3α was deleted from fibroblasts or myofibroblasts with tamoxifen-inducible Tcf21- or periostin- promoter-driven Cre recombinase. In both models, GSK3α deletion restricted pressure overload-induced cardiac fibrosis and preserved cardiac function. We examined the effect of GSK3α deletion on myofibroblast transformation and pro-fibrotic TGFβ1-SMAD3 signaling
in vitro
. A significant reduction in cell migration, collagen gel contraction, and α-SMA expression in TGFβ1-treated KO CFs confirmed that GSK3α is required for myofibroblast transformation. Surprisingly, GSK3α deletion did not affect SMAD3 activation, indicating the pro-fibrotic role of GSK3α is SMAD3 independent. To further delineate the underlying mechanisms, proteins were isolated from CFs of WT and KO mice at 4 weeks post-injury, and kinome profiling was performed. The kinome analysis identified the downregulation of RAF family kinase activity in KO CFs. Moreover, mapping of significantly altered kinases against literature annotated interactions generated ERK-centric networks. Consistently, flow cytometric analysis of CFs confirmed significantly low levels of pERK in KO mice. Additionally, our
in vitro
studies demonstrated that GSK3α deletion prevents TGFβ1-induced ERK activation. Interestingly, IL-11, a pro-fibrotic downstream effector of TGFβ1, was remarkably reduced in KO CFs and ERK inhibition further decreased IL-11 expression. Taken together, herein, we discovered the GSK3α-ERK-IL-11 signaling as a critical pro-fibrotic pathway in the heart. Strategies to inhibit this pro-fibrotic network could prevent adverse fibrosis and HF.

Conclusion:
CF-GSK3α plays a causal role in myocardial fibrosis that could be therapeutically targeted for future clinical applications.
Abstract P320: The Gut Microbial Metabolite Butyrate Suppresses Cardiac Hypertrophy Via An Epigenetic Mechanism
Authors: Umei Masahiko , Akazawa Hiroshi , Saga-Kamo Akiko , Yagi Hiroki , Liu Qing , Kadowaki Hiroshi , Matsuoka Ryo , Shindo Akito , Nomura Seitaro , Komuro Issei
Published: November 2021

Introduction:
Short-chain fatty acids (SCFA) are one of the gut microbial metabolites that can influence host health and disease. We previously reported that gut dysbiosis is associated with heart failure, and that the proportion of butyrate-producing bacteria is decreased in the gut of patients with heart failure.

Purpose:
We investigated the molecular mechanism of butyrate in the development of cardiac hypertrophy.

Methods and Results:
Single-cell transcriptome analysis and co-expression network analysis revealed that G protein-coupled receptors for short-chain fatty acid receptors were not expressed in cardiomyocytes and that Olfr78 was expressed in vascular smooth muscle cells in the heart. On the other hand, treatment with butyrate inhibited ET1-induced and isoproterenol (ISO)-induced hypertrophic growth in cultured neonatal rat cardiomyocytes. Moreover, butyrate increased the acetylation levels of histone H3, suggesting the inhibitory effect of butyrate on HDAC. In addition, butyrate caused the degradation of HDAC2 and up-regulation of Inpp5f, encoding inositol polyphosphate-5-phosphatase f, leading to a significant decrease in the phosphorylation levels of Akt and glycogen synthase kinase 3β (GSK3β). Finally, intraperitoneal injection of butyrate inhibited ISO-induced cardiac hypertrophy in mice. These results suggest that butyrate protects against hypertrophic responses via suppression of the Akt-GSK3β pathway through HDAC inhibition.

Conclusion:
In the heart, there were no known short-chain fatty acid receptors in cardiomyocytes. However, butyrate was shown to have an epigenetic mechanism in suppressing effect on cardiomyocyte hypertrophy via suppression of HDAC2-Akt-GSK3β axis. Our results uncover a potential link between dysbiosis of intestinal microbiota and the development of cardiac hypertrophy.
Abstract P323: Impact Of Diminished Pulsatility On Unravelling Of Von Willebrand Factor (VWF) In Patients With Continuous Flow Ventricular Assisted Devices (CF-VADs)
Authors: Nguyen Khanh T , Wang Yi , Giridharan Guruprasad A , Cheng Xuanhong , Sethu Palaniappan
Published: November 2021

Introduction:
Patients implanted with Continuous Flow Ventricular Assisted Devices (CF VADs) exhibit diminished pulsatility and are at a high risk for developing acquired von Willebrand Factor syndrome (AVWS) and non-surgical bleeding. This study aimed to understand how diminished pulsatility due to CF VAD impacts unravelling and patient plasma levels of von Willebrand Factor (vWF). A microfluidic approach was used to study unravelling of vWF under normal pulsatile flow and flow with diminished pulsatility. In addition, vWF levels in CF-VAD patients was measured to determine vWF levels in circulation.

Hypothesis:
We hypothesized that diminished pulsatility increases vWF unravelling, likely leading to increased vWF degradation and elevated levels of low MW vWF fragments in circulation, this in turn leads to decreased endothelial vWF production in CF-VAD patients.

Methods:
vWF molecules were immobilized in a microfluidic device and subjected to either normal pulsatile flow or flow with diminished pulsatility (same mean flow). vWF unravelling behavior was observed using total internal reflection fluorescence (TIRF) microscopy. Patient blood samples were collected 1-2 days pre CF-VAD implant and monthly post-implant. Patient plasma vWF levels were measured using an ELISA kit.

Results:
TIRF imaging showed that vWF molecules undergo unravelling and significantly greater elongation (p<0.05) under diminished pulsatility than with normal physiological pulsatility, despite higher levels of peak shear rates with normal pulsatility (
Fig.1A
). Evaluation of plasma vWF levels in patients (n=9) showed that vWF levels decreased progressively following CF-VAD placement (
Fig.1B
). These results suggest that diminished pulsatility increased unravelling of vWF and exposure of ADAMTS13 binding sites, potentially leading to enhanced cleavage of vWF into low molecular weight (MW) multimers. Review of literature suggests that both low MW multimers and diminished pulsatility cause endothelial dysfunction and decreased endothelial vWF production, which was evident in patient samples.

Conclusion:
Diminished pulsatility may independently promote vWF degradation and lead to decreased production of vWF, thus contributing to AVWS.
Abstract P324: Brd4 Degradation Enhances Cardiac Reprogramming And Regeneration By Inhibiting JAK/STAT Pathway
Authors: Liu Liu , Guo Yijing , Lei Ienglam , Tian Shuo , Gao Wenbin , Wang Zhong
Published: November 2021

Reprogramming of fibroblasts into induced cardiomyocytes (iCMs) is a potentially promising strategy for regenerating a damaged heart. However, low conversion rate of fibroblasts to cardiomyocytes and poor in vivo application efficacy are major challenges in this reprogramming process. To address this issue, we attempted to identify small molecules related to histone acyl post-translational modifications that could enhance the reprogramming ability towards cardiac fate. Using α-muscle heavy chain-GFP-tagged mouse embryo fibroblasts as a starting cell type, we screened 46 inhibitors target histone acyl post-translational modifications and related epigenetic factors and identified an important role of Brd4 in modulating iCM reprogramming. In particular, we observed that a novel Brd4 degrader repressed many genes involved in immune responses especially JAK/STAT pathway. Mechanically, Brd4 degrader repressed JAK/STAT related gene expressions by affecting Brd4 binding to promoters of those genes. More importantly, Brd4 degrader treatment enhanced MGT induced cardiac regeneration in vivo and markedly improved myocardial performance after myocardial infarction. These findings shed new light on the molecular mechanisms underlying the cardiac conversion of fibroblasts and provide novel targets and small molecules to improve iCM reprogramming for clinical applications.
Abstract P326: Nicotinamide Riboside Kinase-2 Deficiency Promotes Pressure Overload- Induced Dilated Cardiomyopathy And Heart Failure
Authors: Shahzadi Syeda K , Qaisar Rizwan , Ahmad Firdos
Published: November 2021

Dilated cardiomyopathy (DCM) is one of the leading causes of heart failure with a poor prognosis. Nicotinamide riboside kinase-2 (NRK-2), a muscle-specific β1 integrin-binding protein, is predominantly expressed in skeletal muscle and upregulates in several heart failure models. Emerging pieces of evidence suggest that NRK-2 plays a key role in cardiac pathogenesis, however, its role in chronic pressure overload (PO)-induced cardiac remodeling is largely unknown. To investigate the potential role of NRK-2 in PO-induced DCM and delineate the underlying molecular mechanisms,
NRK2
knockout (KO) and littermate control mice were subjected to trans-aortic constriction (TAC) or sham surgeries and cardiac function was assessed by serial M-mode echocardiography. A mild cardiac contractile dysfunction was observed in the KOs at the early adaptive phase of remodeling followed by a significant LV chamber dilatation [LVIDd(mm); 5.32±0.36 vs. 4.75±0.39,
P
=0.001] and functional deterioration [LVEF(%); 20.53±8.81vs. 31.2±7.0,
P
=0.003] during the maladaptive cardiac remodeling phase (6 week). Consistently,
NRK2
KO hearts displayed increased cardiac hypertrophy and heart failure reflected by morphometric parameters as well as increased fetal genes
ANP
and
BNP
expressions. Histological assessment revealed an extensive left ventricular (LV) chamber dilatation accompanied by elevated cardiomyopathy and fibrosis in the KO hearts post-TAC. In a gain-of-function model, NRK-2 overexpressing AC16 cardiomyocytes displayed significantly attenuated fetal genes expression and, NRK-2 further suppressed the fetal gene expression when challenged with angiotensin II. Consistently, NRK-2 overexpression attenuated angiotensin II-induced cardiomyocyte death. Mechanistically, NRK-2 was identified as a critical regulator of JNK MAP kinase and NRK-2 overexpression markedly suppressed the angiotensin II-induced JNK activation. Overall, our results demonstrate that NRK-2 regulates dilatative cardiac remodeling and, genetic ablation exacerbates dilated cardiomyopathy, interstitial collagen deposition, and cardiac dysfunction post-TAC due, in part, to increased JNK activation.
Abstract P332: Microfluidic Model Of Late-stage Calcific Aortic Valve Disease Develops Calcium Phosphate Mineralizations
Authors: Mendoza Melissa , Chen Mei-Hsiu , Murray Bruce , Huang Peter , Mahler Gretchen
Published: November 2021

Introduction:
Calcific aortic valve disease (CAVD) is an active pathological process leading to severe valve calcification. Late-stage CAVD is characterized by increased leaflet stiffness, disorganized collagen bundles and the deposition of glycosaminoglycans, such as chondroitin sulfate (CS), in the fibrosa layer. However, many details of the cellular pathological cascade remain unknown. Animal models such as mice, rabbits, and pigs are used in understanding human CAVD, but mice do not have similar anatomy, rabbits cannot spontaneously develop atherosclerotic lesions, and pigs require long, expensive and complex studies. Here we utilize microfluidic devices of the aortic valve fibrosa to model late-stage CAVD.

Hypothesis:
We assessed the hypothesis that microfluidic calcification will increase with increased shear rates and CS content.

Methods:
Valve-on-a-chip devices contained a hydrogel of 1.5 mg/mL collagen I-only healthy controls or 1.5 mg/mL collagen I with 1 mg/mL or 20 mg/mL CS. Porcine aortic valve interstitial cells (PAVIC) were embedded within and endothelial cells (PAVEC) were seeded onto the matrix. Steady shear stress at 1 dyne/cm
2
and 20 dyne/cm
2
were applied using a peristaltic pump for 14 days. Alizarin Red S (ARS), an assay to assess calcium deposition, was used to quantify calcific nodule formation. Scanning electron microscopy with energy dispersive x-ray (SEM/EDX) was used to further analyze sample mineralization.

Results:
Co-cultures in the presence of increasing shear stress and CS exhibit increased calcific nodule formation compared to static controls, both qualitatively and quantitatively (n≥3). SEM revealed the microstructure of calcified nodules and EDX confirmed calcium phosphate mineralization with physiologically-relevant calcium to phosphorous ratios (Ca/P= 0.88 - 1.4).

Conclusions:
These results show that
in vitro
calcification is driven by shear stress in the presence of PAVEC and CS. As seen in
ex vivo
studies of human calcification, these microfluidic-derived nodules are similarly composed of a range of naturally-occurring calcium phosphates. Given that CAVD has no targeted therapy, the creation of a physiologically relevant model of the aortic valve can provide a test bed for novel therapeutic interventions.
Abstract P334: Biophysical Properties Of A Novel Mitochondrial Large Ubiquitous Non-selective Amiloride Sensitive (luna) Current
Authors: Balderas-Angeles Enrique , Shankar Thirupura , Balynas Anthony , Yin Xue , Chaudhuri Dipayan
Published: November 2021

Inner mitochondrial membrane (IMM) ion channels and transporters account for communication of the matrix with the intermembrane space (IMS) and the cytosol. Transport of solutes and ions is keep under strict regulation mainly because small changes in solute concentrations could generate changes in mitochondrial volume or membrane potential (ΔΨ
m
), interrupting ATP synthesis and leading to mitochondrial damage. The list of recently discovered mitochondrial ion channels has been growing in the past decades. In this work, using the patch-clamp technique we observed the activity of a novel mitochondrial current, named here LUNA current, in mitoplasts (IMM striped of outer membrane) from mouse liver, spleen, brain and heart, as well as established cell lines. LUNA is a novel non-selective cation current (K
+
>Na
+
>NMDG
+
>H
+
) active at depolarized membrane potentials. The basal activity of whole-mitoplast LUNA currents from wild type mice hearts changed from 445±106 pA/pF to 1232±287 pA/pF upon chelation of external divalent cations (Ca
2+
and Mg
2+
). Moreover, the activity of LUNA is independent of the mitochondrial Ca
2+
uniporter and of the non-selective reactive oxygen species modulator channel (ROMO1). In the heart, the activity of LUNA was enhanced in both the
Tfam
-KO mice, which have impaired electron transport chain (ETC) activity and are a model for mitochondrial cardiomyopathies, and mice with cardiac pressure overload due to transverse aortic constriction (TAC) compared to sham-operated hearts (729±197; n=7 vs 283±137 pA/pF). LUNA current is reversibly inhibited by amiloride with no sensitivity to the vast majority of common K
+
, Na
+
and Ca
2+
channels and ETC inhibitors. The molecular identity of mitochondrial LUNA current remains to be determined.
Abstract P335: Nanoparticle-mediated Delivery Of TT-10 Promotes Myocardial Repair
Authors: Chen Wangping , Pretorius Danielle , Zhou Yang , Zhang Jianyi
Published: November 2021

The pharmaceutical product TT-10 increases cardiomyocyte proliferation and survival by upregulating nuclear levels of Yes-associated protein (Yap) and the expression of Yap-targeted genes. When evaluated in a mouse model of myocardial infarction (MI), intraperitoneal TT-10 administration was associated with improvements in infarct size and cardiac function one week after treatment, but functional parameters progressively declined at later time points [1]. Here, we tested our hypothesis that the potency of TT-10 for myocardial repair could be increased by loading it into poly-lactic-co-glycolic acid nanoparticles (NPs) before intramyocardial administration. Mice were treated with injections of DPBS (n=9), empty nanoparticles (Empty-NPs, n=8), TT-10 solution (TT-10-sol, n=8), or TT-10-loaded NPs (TT-10-NPs, n=9) immediately after MI was induced via permanent ligation of the coronary artery. Cardiac function was assessed via transthoracic echocardiography, infarct size via histological Fast-Green staining, cardiac hypertrophy via measures of the heart-weight:bodyweight (HW:BW) ratio, cardiomyocyte proliferation via staining for Ki67 and phosphorylated histone 3 (PH3), and apoptosis via TUNEL. In vitro assessments confirmed that TT-10 was released from the TT-10-NPs for up to four weeks, and that the NPs were taken up by cultured cardiomyocytes. One and four weeks after MI induction and treatment, measures of left-ventricular (LV) ejection fraction (EF) and fractional shortening (FS) were significantly greater in TT-10-NP animals than in the TT-10-sol, Empty-NP, or DPBS groups; LV EF and FS also remained stable in the TT-10-NP group, while measurements in all other groups worsened (but not significantly) (LV EF at Week 4: TT-10-sol vs TT-10-NPs; 22.25 ± 1.474 vs 43.95 ± 1.884, p<0.05), between the two time points. TT-10-NP treatment was also associated with significantly smaller (by ~20%, p<0.05) infarcts and significantly lower HW:BW ratios at Week 4; with significantly greater proportions of Ki67
+
, PH3
+
, and nuclear-Yap
+
cardiomyocytes at Week 1; and with significant declines in TUNEL
+
cardiomyocytes 72 hours after MI and treatment. Thus, NP-mediated delivery of TT-10 results in the efficacy of both the magnitude and durability of the benefits associated with promotion of myocyte proliferation in a mouse MI model.

[1] H. Hara, N. Takeda, M. Kondo, M. Kubota, T. Saito, J. Maruyama, et al., Discovery of a Small Molecule to Increase Cardiomyocytes and Protect the Heart After Ischemic Injury, JACC Basic Transl Sci, 3 (2018) 639-653.
Abstract P336: Endogenous Alpha-1a Adrenergic Receptors Promote Cardiomyocyte Survival Through Suppression Of Rip Kinase-mediated Necroptosis
Authors: Zhang Jiandong , Sandroni Peyton , Huang Wei , Jensen Brian C
Published: November 2021

Our previous work has demonstrated essential protective roles for the endogenous cardiomyocyte alpha-1A adrenergic receptor (α1A-AR) subtype in mouse models of heart failure. However, the underlying mechanism of this protective phenotype is unclear. To address this gap in knowledge, we bred a mouse line lacking α1A-ARs on cardiomyocytes by crossing αMHC-cre mice with floxed α1A mice (CMKO= cre+ fl/fl, CMWT= cre- fl/fl), and subjected males to permanent LAD ligation. CMKO mice had increased serum HMGB1 level, larger infarcts and higher mortality. We found that RIP1/3-mediated programmed necrosis (necroptosis), but not apoptosis was exaggerated in CMKO mice 3 days after ligation. We then tested whether RIP1 inhibition with Nec-1s could mitigate this injury. Mice were given Nec-1s (1.65 mg/kg) or vehicle 10 mins prior to LAD ligation, followed by daily IV injection. Nec-1s treatment diminished post-ligation RIP1 (0.62±0.02 vs. 0.78±0.23 A.U., p=NS) and RIP3 expression (0.33±0.1 vs. 0.26±0.10 A.U., p=NS) in CMWT and CMKO mice respectively. Serum level of HMGB1 on D3 was markedly reduced in both CMWT (45.1%) and CMKO (61.1 %) after Nec-1s treatment. There was no difference between Nec-1s treated CMWT and CMKO mice (147±53 vs. 174±37 pg/mL, p=NS), indicating that blocking the RIP kinase pathway abrogates the exaggerated cell death in CMKO mice after ligation. Likewise, Nec-1s-treated CMKO mice had similar infarct areas to CMWT controls (16.2±4.5 vs. 19.9±4.6%, p=NS), further confirming that targeting necroptosis abrogates pathological damage. Collectively these Nec-1s data suggest that RIP-mediated necroptosis may account for larger infarcts in CMKO mice. Interestingly, expression of the apoptosis markers c-caspase-3 and PARP was similar between CMWT and CMKO mice, suggesting that the α1A-AR specifically regulates necroptosis. In sum, our data demonstrate that RIP kinase-mediated necroptosis contributes to susceptibility to injury in mice lacking cardiomyocyte α1A-ARs.
Abstract P352: An Antiplatelet Agent Sarpogrelate Suppresses Pressure Overload-induced Development Of Heart Failure In A 5-ht
2a
Receptor-independent Manner
Authors: Shimizu Kana , Funamoto Masafumi , Sunagawa Yoichi , Katanasaka Yasufumi , Miyazaki Yusuke , Murai Noriyuki , Shimizu Satoshi , Hasegawa Koji , Morimoto Tatsuya
Published: November 2021

Purpose:
The cost of new drug development is increasing year by year, and drug repositioning is being used as a strategy to develop new treatments at low-cost. We used a library of approved drugs to screen for compounds that suppress cardiomyocyte hypertrophy, and identified as a candidate the antiplatelet drug sarpogrelate, a selective serotonin-2A (5-HT
2A
) receptor antagonist. In this study, we examined the effect of sarpogrelate on cultured cardiomyocyte hypertrophy and development of heart failure.

Methods & Results:
First, primary cultured cardiomyocytes were treated with 1 μM sarpogrelate and then stimulated with various hypertrophic stimuli (30 μM phenylephrine (PE), 0.1 μM angiotensin II and 0.1 μM endothelin 1). The results of immunofluorescence staining with anti-MHC antibody showed that sarpogrelate significantly suppressed cardiomyocyte hypertrophy induced by each stimulus. Western blotting and qPCR analysis showed that the mRNA and protein levels of 5-HT
2A
receptor did not change by PE, and sarpogrelate significantly suppressed PE-induced phosphorylation of ERK1/2 and GATA4. Next, C57BL/6j male mice were subjected to a transverse aortic constriction (TAC) and sham operation. One day after the operation, the mice were randomly divided into 3 groups: sarpogrelate at 1 mg/kg or 5 mg/kg, and vehicle as a control. Daily oral administration was repeated for 8 weeks. Echocardiographic analysis showed that 5 mg/kg sarpogrelate significantly prevented a TAC-induced increase in posterior left ventricular wall thickness and a decrease in fractional shortening at 8 weeks after the operation. Five mg/kg sarpogrelate also suppressed TAC-induced increase in HW/BW ratio, cross-sectional areas, perivascular fibrosis, and mRNA levels of ANF and BNP. Moreover, the western blotting analysis showed that 5 mg/kg sarpogrelate significantly suppressed TAC-induced phosphorylation of ERK1/2.

Conclusions:
These results indicate that sarpogrelate significantly suppresses cardiomyocyte hypertrophy and the development of heart failure via at least, in part, by inhibition of ERK1/2-GATA4 pathway. These findings suggest that sarpogrelate may be an effective agent for heart failure therapy.
Abstract P353: Adipose-derived Stem Cells Contribute To Vascular Remodeling Via
Clec11a
+ Subpopulation
Authors: Ji Yongli , Lu Yunrui , Shen Jian , Xiang Meixiang , Xie Yao
Published: November 2021

Introduction:
Recent researches identified the existence of perivascular adipose-derived stem cells (ADSCs) which could differentiate into vascular lineages and participate in vascular remodeling. Single-cell mRNA analysis revealed cellular heterogeneity of subcutaneous ADSCs in respect to cell clustering and cell differentiation. However, such analysis of perivascular ADSCs has not been investigated at a single-cell level.

Hypothesis:
There is a significant difference among perivascular ADSCs subpopulations in respect to vascular-lineage differentiation.

Methods:
We performed droplet-based single-cell profiling of subcutaneous and perivascular adipose stromal cells and compared ADSCs regarding their heterogeneity, gene ontology, and cell fate trajectory by applying single-cell analysis as well as
in vitro
and
in vivo
assays.

Results:
Single-cell analysis uncovered 4 perivascular ADSCs subpopulations including
Dpp4+
,
Col4a2+
/
Icam1+
,
Clec11a+
/
Cpe+
and
Sult1e1+
cells. Notably, the
Clec11a
+ subpopulation comprised the bulk of perivascular ADSCs, while was hardly presented in subcutaneous ADSCs. Further gene-set enrichment analysis suggested
Clec11a
+ ADSCs were potentially involved with TGF-β signaling pathways and pseudotemporal analysis predicted that
Clec11a
+ subpopulation lay at the end of the differential trajectory towards smooth muscle cells (SMCs).
In vitro
assays displayed that perivascular ADSCs could differentiate into SMCs via CLEC11A regulation when treated by TGF-β1. To further elucidate the role of the
Clec11a
+ subpopulation in SMCs differentiation, we labeled CLEC11A+ and CLEC11A- perivascular ADSCs by lentivirus transfection and isolated them by FACS assay. CLEC11A+ cells showed the greater capability of SMCs differentiation in response to TGF-β1
in vitro
and enhanced neointima formation when transplanted to the adventitial side of guidewire injured arteries.

Conclusions:
The present study depicted the unique heterogeneity of perivascular ADSCs and the novel role of the
Clec11a
+ subpopulation, providing a supplement for the relationship between perivascular ADSCs and vascular SMCs.