bluuebunny/crossref_metadata_2025 huggingface dataset:
Research on a Power Grid Cascading Failure Prevention and Control Method considering WSN
Authors: Deng Huiqiong , Wu Junyuan , Luo Jie , Yan Renwu , Zhang Cheng , Li Peiqiang , Chang Kuo-Chi , Zheng Rongjin
Published: November 2021

The practical application of wireless sensor networks (WSNs) in hot fields is summarized. It is found that compared with traditional monitoring methods, it has better adaptability to complex environments and low cost. It is suitable for monitoring power grid operation parameters. Therefore, this paper combines the above network and cascading failures, analyzes its 24‐hour continuous and dynamic monitoring of the operation parameters of the power grid, and considers how to use the obtained parameters to analyze the disturbance of the remaining lines after the initial fault of the power grid. To prevent cascading failures in the power grid, a preventive control model considering safety and economy is proposed, and the model is solved by nondominated sorting genetic algorithm II and particle swarm optimization (NSGA2‐PSO). Finally, the rationality of this method is verified in the IEEE39 node system.
Autopsy Findings of Heterozygous Fabry Disease with the Severe Phenotype: A Case Report
Authors: Hiratsuka Maki , Koyama Katsushi , Ito Makoto , Sato Ryo , Suzuki Kodai , Ito Yuki , Namba-Hamano Tomoko , Hamano Takayuki
Published: November 2021

Fabry disease (FD) is an inherited X-linked lysosomal storage disorder, with hemizygous males being more severely affected than heterozygous females. Herein, we report a rare case of FD in a heterozygous female with a severe phenotype. The patient had obesity and hyperlipidemia and had her first cerebral infarction at the age of 33 years. She underwent renal biopsy and was diagnosed with FD with morphological features of focal segmental glomerulosclerosis nephropathy at the age of 34 years. Her leukocyte alpha-galactosidase A activity was 2.3 Agal/U (normal: >20 Agal/U), and genetic analysis revealed the presence of the classical phenotype. Enzyme replacement therapy (ERT) was initiated at the age of 35 years; however, peritoneal dialysis owing to end-stage renal failure occurred at the age of 37 years. The patient died of a cerebral hemorrhage at the age of 44 years. Her Mainz Severity Score Index at the time of death was 48/76, suggestive of the severe phenotype. Autopsy findings revealed remarkable globotriaosylceramide accumulation on electron microscopy, particularly in the major organs and their vascular smooth muscle cells. Regarding the vertebral arteries which sourced the cerebral hemorrhage, the effects of FD-induced vascular thickening and long-term renal failure-induced atherosclerosis were confirmed. Furthermore, the patient’s vascular sclerosis was modified with acquired factors such as lifestyle-related disease associated with obesity. We recommend intensified treatment for metabolic factors simultaneous with ERT to help in delaying the progression of FD.
Abstract 102: Time-of-intake Regulates Glucocorticoid Pharmacology Of Cardiac Bioenergetics
Authors: Quattrocelli Mattia , Wintzinger Michelle , Miz Karen
Published: November 2021

Glucocorticoid steroids are circadian regulators of energy balance. However, the specific direct effects of glucocorticoids on heart metabolism remain unresolved. Moreover, the impact of circadian time-of-intake on glucocorticoid pharmacology is still unknown. Here, we investigated whether circadian time of exposure gates the effects of synthetic glucocorticoids on heart bioenergetics. We compared the effects of diurnal versus nocturnal glucocorticoids in heart tissue and mitochondria from wildtype mice, controlling the subjective circadian time of drug injection. To avoid interferences from other tissues, we developed an ex vivo system to interrogate the mitochondrial respiratory capacity rate (state III/state IV) in isolated hearts. We found that diurnal but not nocturnal pulse of the glucocorticoid prednisone increased the mitochondrial respiratory capacity rate in heart. This correlated with circadian-restricted effects on mitochondrial abundance. This was remarkable as it contrasts the circadian fluctuations of endogenous glucocorticoids. Using transgenic mice with inducible cardiac-specific gene knockout, we found that the bioenergetic effects of diurnal-restricted prednisone were dependent on the glucocorticoid receptor and its co-factor Kruppel-like factor 15. Considering the bioenergetic decline that hallmarks the aging heart, we asked whether these circadian-gated effects were applicable to aged mice. We therefore treated 24 months-old mice for 12 weeks with a diurnal-restricted regimen of prednisone. Compared to vehicle, diurnal prednisone increased mitochondrial respiration along with NAD
+
and ATP content in aged hearts. Moreover, lipidomic profiling of myocardial tissue showed that the vast majority of lipids were downregulated after treatment, including triacylglycerols, suggesting a functional coupling between lipid utilization and mitochondrial oxidation in treated hearts. We also found that diurnal-restricted prednisone rescued bioenergetics and improved function in diabetic hearts from db/db mice. In summary, our data indicate that glucocorticoids regulate cardiac bioenergetics according to circadian-time of intake, supporting a role for chrono-pharmacology in aged and diabetic hearts.
Abstract 107: Selective Igf-1 Production By Resident Cardiac Macrophages Orchestrates Adaptive Cardiomyocyte Growth During Hypertensive Stress
Authors: Zaman Rysa , Hamidzada Homaira , Epelman Slava
Published: November 2021

Objective:
Adaptive cardiomyocyte growth is an essential compensatory response to hypertension. While hypertension activates cardiac immune cells, their role in adaptation is unclear. Here, we define the transcriptional heterogeneity and functional role of cardiac resident macrophages (RMs)
in vivo
in hypertensive heart disease.

Methods:
We performed flow cytometry, immunofluorescence, and single-cell transcriptomics on fate-mapped cardiac RMs isolated from mice with angiotensin II infusion in acute (day 4) and chronic hypertensive stress (day 28). Following inducible depletion of RMs or specific genetic deletion of
Igf1
in RMs during hypertension in mice, functional outcomes were tested with echocardiography and immunohistochemistry. Lastly, we also performed single-cell RNA sequencing on human cardiac macrophages from healthy and diseased samples.

Results:
Cardiac RMs possess numerous transcriptionally diverse cell states with a core repertoire of reparative gene programs that includes high expression of
Igf1
in normotensive animals. Individual cell states were differentially responsive during hypertension while all maintained their original transcriptional identity. Hypertension drove selective
in situ
proliferation and numerical expansion of some cardiac RMs, directly correlating with increased cardiomyocyte size. Inducible ablation of RMs, or selective deletion of RM-derived IGF-1 caused complete absence of adaptive cardiomyocyte growth and development of cardiac dysfunction. Single-cell transcriptomics further identified a conserved
IGF1
-expressing macrophage subpopulation in human cardiomyopathy.

Conclusions:
Here, we defined the absolute requirement of cardiac RM-produced IGF-1 in adaptive cardiomyocyte growth during hypertension, identifying a novel and essential pathway of RM-directed cardiac adaptation to disease.
Abstract 115: Cell-type-specific Gene Expression And Transcriptional Networks Reveal
Adamts2
As A Powerful Regulator Of Cardiac Homeostasis During Heart Failure
Authors: Lahue Caitlin , Chapski Douglas , Rosa Garrido Manuel , Ren Shuxun , Vondriska Thomas M , Wang Yibin , Rau Christoph
Published: November 2021

Background:
Heart failure (HF) is a highly heterogeneous disorder characterized by the interactions of multiple genetic and environmental factors as well as the interaction of different cell types in the heart. Although reductionistic approaches have successfully identified many genes involved in HF, heritability studies suggest that many genes have resisted discovery through these approaches. By utilizing cell-type-specific gene expression paired with transcriptomic data from a large cohort of mice, we sought to identify important drivers of HF using a systems genetics approach.

Methods and Results:
Mice from 93 unique inbred lines of the Hybrid Mouse Diversity Panel were given 30 ug/g/day of isoproterenol for three weeks via osmotic minipump to induce heart failure. Transcriptomes were generated from these mice and the weighted Maximal Information Component Analysis (wMICA) algorithm was applied to generate transcriptomic gene networks. Cardiomyocytes and Fibroblasts were isolated from both control and isoproterenol-treated adult C57BL/6J hearts using a Langendorff apparatus (n=3 per sex/treatment) and transcriptomes were generated. Significantly differentially expressed genes were identified using DESEQ2 and used to query the wMICA-derived network, identifying the gene
Adamts2
as a potential regulator of cardiac hypertrophy. Follow-up
in vitro
and
in vivo
work has demonstrated that
Adamts2
knockdown significantly blunts the hypertrophic effect of isoproterenol on cardiomyocytes while simultaneously reducing fibroblast proliferation and increasing apoptosis as measured by TUNEL staining. Careful examination of the gene network reveals evidence of paracrine signaling between cardiomyocytes and fibroblasts and suggests a key trans-cell-type role of
Adamts2
in the regulation of HF after catecholamine stimulation.

Conclusion:
Co-expression network algorithms combined with cell-type-specific transcriptomics identified
Adamts2
as a driver of HF.
Adamts2
plays an important role via paracrine signaling in the proliferative response of fibroblasts and the hypertrophic response of cardiomyocytes to catecholamines. Further mechanistic analysis of
Adamts2
will further reveal its role in the progression of heart failure.
Abstract 118: Interleukin 4 And 13 Signaling In Macrophages Regulates Neonatal Cardiac Regeneration
Authors: Alvarez Santiago , Omeara Caitlin
Published: November 2021

Introduction:
Heart failure (HF) is a prevalent disease, projected to affect over 8 million Americans by 2030. Current therapy partially decreases progression; however, mortality and disease burden continue to be high. Therefore, there is an unmet need to develop new strategies that target HF progression. Our lab employs the neonatal mouse model of cardiac regeneration to identify pro-reparative pathways that can be applied to treating HF in humans. Previously we demonstrated that the anti-inflammatory cytokine, Interleukin 13 (IL13), promotes cardiac regeneration, however, the cell types mediating this response remain unknown. IL13 and the related cytokine, IL4, share a common receptor (IL4Rα) and both cytokines polarize macrophages into a reparative phenotype. Here, we hypothesize that IL4/13 signaling to macrophages promotes heart regeneration after cardiac injury and we explore the cell source of these cytokines during neonatal development and cardiac regeneration.

Methods and Results:
We generated a genetic model whereby IL4Rα is depleted in macrophages by crossing IL4Rα floxed (IL4Rα
fl/fl
) mice with transgenic mice CX3Cr1 driven-Cre recombinase (CX3CR1
Cre
). Flow cytometry analysis confirmed depletion of IL4Rα in cardiac macrophages. We performed myocardial infarction (MI) on postnatal day 1 (P1) mice and assessed cardiac regeneration by ultrasonography 21 days post-injury (dpi). We found that IL4Rα
fl/fl
CX3CR1
Cre
mice had lower ejection fraction compared to IL4Rα
fl/fl
littermate controls. Preliminary results suggest there is a reduced capillary density in peri-ischemic myocardium. In addition, we used fluorescent reporter mouse lines; IL4-enhanced green fluorescent protein (IL4-GFP) and IL13-yellow fluorescent protein (IL13-YFP), to assess the cellular source of IL4 and IL13 expression in the hearts of neonatal mice. In unoperated mice, we found no detectable expression of IL13 by any cell type in the heart, whereas IL4 was expressed in innate lymphoid cells (ILCs) and T cells. 4 days after MI in P1 mice IL13 was upregulated in ILCs and T cells and IL4 was expressed by ILCs and T cells and upregulated in myeloid cells.

Conclusions and Discussion:
We found that IL13 and IL4 are primarily expressed in ILCs and T cells following neonatal injury, suggesting a novel role for ILCs and T cells in the production of IL4 and 13 during the neonatal regeneration process. In addition, we found that lack of IL4/13 signaling in macrophages via depletion IL4Rα impairs cardiac regeneration after MI in neonatal mice, and results in reduced capillary density in peri-ischemic myocardium. We hypothesize that IL4Rα depletion in macrophages impairs reparative macrophage polarization after MI and promotes an inflammatory polarization. Future studies will be aimed to assess macrophage phenotypes in response to IL13/IL4 signaling by transcriptional profiling and flow cytometry.
Abstract 120: Pericytes Contribute To Myofibroblast Expansion And Vascular Maturation In The Infarcted Heart
Authors: Alex Linda , Su Kai , Tuleta Izabela , Frangogiannis Nikolaos G
Published: November 2021

Infarct healing is dependent on recruitment of inflammatory leukocytes and subsequent activation of myofibroblasts (MF) and neovessel formation, ultimately resulting in formation of a highly vascularized collagen-enriched scar. Though the heart has an abundant population of periendothelial pericytes, its role in wound healing upon myocardial infarction (MI) has not been studied. We hypothesized that in the infarcted myocardium, pericytes may become activated, contributing to inflammatory, fibrotic and angiogenic responses. We used pericyte/fibroblast reporter mice (NG2
DsRed
;PDGFRα
GFP
), lineage tracing studies and in vitro approaches to study the fate and role of pericytes in the infarcted myocardium. In normal hearts, NG2+/PDGFRα- pericytes and PDGFRα+/NG2- fibroblasts had distinct transcriptomic profiles. Pericytes expressed mural genes like
Acta2
,
Pdgfrb
and low amounts of extracellular matrix (ECM) genes, whereas fibroblasts synthesized collagens,
Timp2/3
and matricellular genes. 7 days post-MI, expansion of the NG2+ population in the infarct zone was associated with emergence of non-mural NG2+/αSMA+ cells with MF characteristics. FACS-sorted NG2+/PDGFRα- cells from 7-day infarcts expressed higher levels of collagens when compared to NG2+/PDGFRα- cells from normal hearts. Infarct pericytes had high integrin and MMP14 expression, reflecting an activated migratory phenotype. Lineage tracing using NG2CreER
TM
;Rosa
tdTomato
;PDGFRα
GFP
mice showed that 5.7%±1.04 of PDGFRα+ fibroblasts and 10.49%±2.73 of infarct MFs were derived from NG2+ lineage. Pericyte-derived fibroblasts exhibited higher ECM gene synthesis, in comparison to fibroblasts from non-pericyte origin, while pericyte-derived mural cells showed accentuated inflammatory cytokine gene expression. Immunostaining showed pericytes actively contribute to vascular maturation, forming a mural cell coat enwrapping infarct neovessels. In vitro, TGFβ induced integrins, collagens and MMPs in human pericytes, similar to the changes observed in infarct pericytes. Taken together, our evidences show that after MI, pericytes become activated and contribute to repair by undergoing conversion to a subset of myofibroblasts and by coating infarct neovessels.
Abstract 121: Interleukin 11 Mediates Wnt/β-catenin-dependent Fibrotic Response Of Human Cardiac Fibroblasts
Authors: Dzialo Edyta , Czepiel Marcin , Siedlar Maciej , Kania Gabriela , Blyszczuk Przemyslaw
Published: November 2021

Wnt proteins family represents secreted glycoproteins implicated in the number of fibrotic cardiac pathologies. The transcriptional activity of Wnts is broad and involves β-catenin-dependent or β-catenin-independent responses. In this study, we examined the effect of exogenous Wnt3a (β-catenin-dependent) and Wnt5a (β-catenin-independent) in TGF-β-activated human cardiac fibroblasts. Furthermore, we assessed the hypothesis that Wnt3a could regulate IL-11 production and analyzed its contribution to profibrotic response in cardiac fibroblasts.By employing a full genome transcriptomics, we analyzed transformation of human cardiac fibroblasts induced by TGF-β in the presence of Wnt3a or Wnt5a produced by cell culture supernatant of L-Wnt3a, L-Wnt5a or control L-cells. Stimulation with Wnt3a of TGF-β-activated fibroblasts resulted in induction of 66 genes, specifically involved in myofibroblast differentiation including
ACTA2
(encoding alpha smooth muscle actin; αSMA)
ACTG2
(encoding gamma smooth muscle actin; γSMA) and
VCL
(encoding vinculin). In contrast to Wnt3a, treatment with Wnt5a upregulated expression of only 2 genes in TGF-β-activated cells. Additionally, in the presence of TGF-β, Wnt3a enhanced phosphorylation of TAK1 and production and secretion of IL-11. Importantly, in the absence of TGF-β, Wnt3a did not promote fibroblast-to-myofibroblast transition, TAK1 phosphorylation and IL-11 production. To determine, if Wnt3a-dependent production of IL-11 could contribute to profibrotic response we blocked IL-11 activity with anti-IL-11 neutralizing antibody in cardiac fibroblasts activated with TGF-β and Wnt3a. We found that neutralizing anti-IL11 antibody effectively suppressed production of αSMA, γSMA, fibronectin and pro-collagen I alpha 1, both on mRNA and protein levels. In line with these findings, blockade of IL-11 suppressed contractile properties of TGF-β/Wnt3a-activated cardiac fibroblasts. In conclusion, Wnt3a and Wnt5a differentially regulate gene expression of TGF-β-activated cardiac. Activation of the Wnt/β-catenin pathway promotes fibroblast-to-myofibroblast transition by enhancing production of profibrotic IL-11. It seems that identifying the profibrotic Wnt/β-catenin-IL11 mechanism in cardiac fibroblasts might represent a promising strategy in development of new therapies against cardiac fibrosis.
Abstract 402: Defining Diverse Disease Pathomechanisms Across Thick And Thin Filament Hypertrophic Cardiomyopathy Variants.
Authors: Toepfer Christopher , Psaras Yiangos , Margara Francesca , Schmid Manuel , Steeples Violetta , Marsiglia Julia D , Garfinkel Amanda , Repetti Giuliana , Alfonso Bueno-Orovio , Rodriguez Blanca , Seidman Jonathan , Seidman Christine E
Published: November 2021

Hypertrophic cardiomyopathy (HCM) affects as many as ~1 in 500 individuals, and is often typified by hyperdynamic contraction and poor cellular relaxation. HCM can be caused by mutations in a variety of key contractile proteins of the sarcomere. A large proportion of these variants are found in MYBPC3, MYH7, TNNT2, and TNNI3. These genes encode proteins that control cardiac muscle contraction at the thick (MYBPC3 and MYH7) and thin filaments (TNNT2 and TNNI3) of the sarcomere. In this study we use human induced pluripotent stem cell derived cardiomyocytes to model HCM across all of these genes. We do this to define key mechanistic differences between thick and thin filament HCM. We define sarcomeric contractility (SarcTrack) calcium transients (CalTrack) and myosin states using the mant-ATP assay. We use the parametric data from these experimental studies in iPSC-CMs to model possible disease mechanisms in silico. Our experimental analysis highlights that both thick and thin filament HCM variants cause cellular hypercontractility, with slowed cellular relaxation. We find that thick filament HCM variants drive cellular HCM phenotypes by destabilising the myosin interacting heads motif (IHM), showing a marked reduction in the super relaxed state of myosin. Counterintuitively thin filament based HCM variants show a reduction in DRX myosin. When applying Mavacamten the allosteric myosin ATPase inhibitor to our thin and thick filament HCM variant iPSC-CMs we find a dichotomy of cellular responses. The thick filament variants studied all show a clear resolution of cellular HCM. However, not all cellular phenotypes of thin filament HCM are corrected by Mavacamten treatment, although there is benefit. We conclude that causal mechanisms of thick filament HCM are well corrected at the molecular and cellular level by Mavacamten, but these causal mechanisms in thin filament based HCM are not suitably corrected. We highlight key mechanistic pharmacological targets for thin filament variants that could add cellular benefit to HCM phenotype resolution.
Abstract MP202: The Effects Of Maternal Hypothyroidism On Fetal And Adult Cardiac Function
Authors: Li Yijia , Chen Guihong , Zhang Xiaoying , Zhao Ran , Houser Steven R , Chen Xiongwen
Published: November 2021

Objectives:
Maternal hypothyroidism (MH), a common clinical condition with a reported prevalence from 2.5% to 13%. MH could have an adverse effect on fetal cardiac development and adult heart function. However, there is a lack of systematic research for its effect on fetal and offspring’s development and cardiac function. This study aimed to determine the impact of MH on fetal and offspring’s organ structural and functional development at different gestation stages and during postnatal and puberty.

Methods:
MH model had been built through thyroidectomy (TX) with total thyroxine (TT4) under 1ng/dl after surgery. Pups from mice that underwent TX and Sham surgery were named THD (Deficient) and THN (Normal). Times of parturition and miscarriage from TX and Sham groups were recorded. Ultrasound was performed for fetuses and/or offspring to check the gestation, miscarriage, embryo arrest, development, malformation, and cardiac function. At different postnatal days, mice were euthanized for organ development evaluation.

Result:
TX mice had reduced parturition frequencies and smaller litter size but increased miscarriage frequency and malformed fetuses than the sham group. In addition, THD fetuses had smaller biparietal diameter (BPD) and abdominal circumference (AC) and significantly lower left ventricular ejection fraction (LVEF) on E14.5, but not at E18.5. Tei index, a parameter representing both systolic and diastolic cardiac function, was lower in THD fetuses than in THN fetuses at both E14.5 and E18.5. The cardiac systolic and diastolic function of female TX mice after a 9-month breeding period was also abnormal. The Postnatal T4 level was not significantly different between the two groups. On and after d21 after birth, THD mice had a higher heart weight/body weight ratio and lower LVEF still d21.

Conclusion:
MH impaired the development and cardiac function of the fetus and young adult. In addition, MH impaired the mother's fertility and depressed cardiac function.
Abstract MP206: Cortical Bone Derived Stem Cells Modulate The Adaptive Immune Response Post-MI
Authors: Wagner Marcus J , Kraus Lindsay , Kino Tabito , Khan Mohsin , Mohsin Sadia
Published: November 2021

Background:
The inflammatory response mounted following myocardial infarction (MI) is closely coupled to myocardial wound healing processes such as angiogenesis, fibroblast maturation, and cardiomyocyte survival. A consortium of pro-reparative Tregs occupy the myocardium during ischemic injury and are essential in modulating cardiac homeostasis. However, Treg exposure to chronic, ischemic microenvironments compromises Tregs’ pro-reparative signatures and induces pathogenic remodeling of Tregs, compromising cardiac wound healing. Cortical Bone Derived Stem Cells (CBSCs) have superior engraftment capabilities compared to other stem cell types and can modulate the post-MI inflammatory response by establishing anti-inflammatory paracrine signaling dominance and the expansion of CD45+ leukocytes and CD4+ T cells residence in the MI heart. Therefore, we hypothesized CBSC cell therapy can modulate the recruitment and phenotype of Treg residence in the MI heart to permit their enhanced cardiogenic properties.

Methods and Results:
Animals that received intramyocardial injection of CBSCs along the infarct border zone of the permanently ligated LAD possessed smaller infarct sizes and improved cardiac function, that paralleled pro-reparative, TNFRII+ Treg expansion and repressed pathogenic TNFRI+, Treg residence 1- and 8- weeks post-MI compared to Mesenchymal Stem Cells (MSCs) or vehicle (PBS) treated animals. Similar TNFRI/II Treg dynamics were also observed in the spleen. Diphtheria toxin (DTR) mediated Treg ablation and S1P1 agonist administration, in the presence of CBSC therapy, reverted pro-reparative Treg expansion and solicited infarct size expansion and compromised cardiac function. The exposure of CBSC paracrine secretome to naïve CD4+ T cell cultures induces pro-reparative TNFRII+ Treg expansion. Paracrine profiling of CBSC secretome identifies Osteoprotegerin (OPG) enrichment and OPG depletion, via siRNA and lentivirus, compromises T cell survival signaling and pro-reparative, TNFRII+ Treg establishment both
in vitro
and in the MI heart.

Conclusions:
CBSCs can modulate the induction and preservation of pro-reparative TNFRII+ Tregs in the MI heart to solicit cardiac repair during acute and chronic ischemic injury.
Abstract MP207: A Precision Medicine Approach For Non-invasive, Longitudinal, And Quantitative Monitoring Of Cardiac Tissue-engineered Scaffolds
Authors: Gil Carmen , Evans Connor , Li Lan , Vargas Merlyn , Kabboul Gabriella , Fulton Travis , Veneziano Remi , Nick Nick , Bauser-Heaton Holly , Roeder Ryan K , Serpooshan Vahid
Published: November 2021

3D bioprinting has revolutionized personalized and precision medicine by enabling the manufacturing of tissue constructs that precisely recapitulate the cellular and functional features of native tissues. In cardiac regenerative medicine, printed scaffolds have shown tremendous potential in repairing damaged heart, however, their clinical applications have been limited by the lack of precise noninvasive tools to monitor the patch function following implantation. By integrating state-of-the-art 3D bioprinting and photon-counting computed tomography (PCCT), this study introduces a new approach for bioengineering defect-specific scaffolds and monitoring their function. We prepared distinct CT-visible bioinks containing a variety of molecular or nanoparticle (NP) contrast agents, including iodine and gadolinium molecules, Au NPs, Gd
2
O
3
NPs, and iodine-loaded liposomes (
Fig 1A-B
).

In vitro
release experiments showed relatively rapid diffusion-controlled depletion of molecular contrast agents from scaffolds. In contrast, NP agents showed more stable encapsulation and only a partial, degradation-mediated release for up to 3 weeks of incubation (
Fig 1C-D
). Next, PCCT imaging was performed on various scaffold geometries printed using bioinks laden with Gd
2
O
3
or Au NPs. Results demonstrated CT visibility with differential contrast between different patch regions that corresponded to the designed geometries (
Fig 1E
). Finally, we evaluated the
in vivo
CT imaging of bioprinted patches after their subcutaneous implantation in a mouse model. CT images demonstrated adequate signal from implanted grafts (
Fig 1F
). Together, these results establish a novel precision medicine platform for non-invasive monitoring of medical devices which can open new prospects for a broad range of tissue engineering applications.

Figure 1.
3D Bioprinting of CT-visible cardiac patches.
A-B:
Design of bioinks functionalized with molecular (left) and nanoparticle (right) CT contrast agents (
A
) and their bioprinting (
B
).
C-D:
In vitro
release of contrast agents from printed patches.
E:
CAD design (left), CT image (middle), and PCCT material decomposition (right) for multi-contrast bioprinted scaffolds.
F:
In vivo
CT imaging of printed patch, laden with Au NPs, implanted subcutaneously into a mouse torso.
Abstract MP215: Endothelial Colony Forming Cell Derived Exosomes Promote Angiogenesis And Cardiac Repair Post Myocardial Infarction
Authors: Gomes Karina P , Jadli Anshul S , Parasor Ananya , Ballasy Noura N , Surti Monica , Belke Darrell D , Wijesuriya Methsala , Fedak Paul W , Patel Vaibhav B
Published: November 2021

Background:
Despite improvements in therapeutics, ischemic heart disease remains a leading cause of death. Cardiac remodeling after myocardial infarction (MI), predominantly due to loss of cardiomyocytes and coronary vasculature, leads to a progressive decline in cardiac function resulting in heart failure. Current therapies for cardiac repair and heart failure are of limited benefit. Cell transplantation therapy upon MI is a very promising therapeutic strategy to replace dead myocardium, reducing scarring and improving cardiac performance.

Methods and Results:
Our research focuses on endothelial colony-forming cell-derived exosomes (ECFC-exosomes), which are actively secreted endocytic nanovesicles (30-100 nm) that transport functional miRNAs, proteins, mRNAs, and lipids, playing a key role in paracrine intercellular communication. We identified a novel ability of ECFC-exosomes to promote angiogenesis and cardiac tissue repair. Administration of ECFCs to mice following experimental end-organ ischemia resulted in ECFC-exosome-dependent increase in angiogenesis. ECFC-derived exosomes were taken up by endothelial cells leading to their proliferation and migration, tube formation, and formation of new vessels. Administration of ECFC-exosome to a murine model of MI prevented cardiac remodeling and heart failure. The acute MI resulted in severely decreased left ventricle ejection fraction (Sham 71.2% ± 5 .87, MI+Saline 32.9% ± 2.32) and fractional shortening (Sham 29.5% ± 3.20, MI+Saline 13.6% ± 2.87), and the administration of ECFC-exosomes prevented MI-induced cardiac dysfunction (ejection fraction: MI+ECFC-Exo 64.3% ± 8.74; fractional shortening: MI+ECFC-Exo: 26.4% ± 3.13). Next generation sequencing and bioinformatics analyses identified 136 miRNAs present in ECFC-exosome cargo, and factor inhibiting HIF-1α and PTEN as their potential targets in endothelial cells. Increased nuclear HIF-1α levels in response to ECFC-exosome administration, which may aid in the transcriptional function of HIF-1α, corroborated the role of exosomal miRNA in myocardial angiogenesis. We also found decreased levels of PTEN in response to ECFC-exosome treatment, which is a key negative regulator of PI3K/Akt pathways, survival pathways of heart. We also identified the relative angiogenesis expression profile of the peri-infarcted area in response to ECFC-exosome treatment. The ECFC-exosome administration upregulated the levels of VEGF, IGFBP-1 and PDGF, among others proangiogenic factors, and downregulated the levels of angiostatic factors as IP-10 and Thrombospondin-2.

Conclusion:
Our findings support the view that the ECFC-exosomes represent a novel therapeutic approach to improve cardiac repair after MI.
Abstract MP220: Identification And Functional Characterization Of Exosomal Bk Channels
Authors: Sanghvi Shridhar , Dougherty Julie A , Evans Parker , Sridharan Divya , Ponnalagu Devasena , Szteyn Kalina , Sundaresan Vishnu , Khan Mahmood , Singh Harpreet
Published: November 2021

Extracellular vesicles (EVs) specifically exosomes are important in mediating intracellular communications, and are capable of transferring genetic information between cells. Exosomes are used as a drug delivery vehicle to carry cargo for targeted therapy and have emerged as one of the most promising candidate for treating cardiovascular diseases. Exosomes get packaged inside the cell, excise out to the extracellular environment, and deliver the cargo to the target cells. However, the precise mechanism of how exosomes handle the differential ionic environment and the physiological role of their ion channels is not determined. Given that potassium (K
+
) ions has the largest gradient, we focused on identifying the presence and physiological relevance of K+ channels in exosomes. Using the
in silico
approach, several ion channel candidates were identified, the most prominent ion channel being large conductance Ca
2+
and voltage-activated potassium channel (BK). To record BK in exosomes, we incorporated planar bilayers and a novel electrophysiology approach called near field electrophysiology (NFE), as the canonical patch-clamp methods are not feasible due to the size of EVs. Our NFE indicates a presence of K
+
channels in intact exosomes and 45% of them are sensitive to IbTX. Since IbTX specifically blocks, BK channels, we estimated 2 functional channels (single-channel conductance of 300 pS with 50% open probability) in a single exosome. Plasma-derived exosomes from BK
+/+
and BK
-/-
mice subjected to differential K
+
gradient indicated that functional BK channels exist in exosomes, and help in maintaining their structural integrity. Furthermore, plasma derived exosomes from BK
+/+
mice showed cardioprotection from ischemia-reperfusion injury whereas exosomes from BK
-/-
mice did not. Thus, the presence of BK determines the packaging as well as cardioprotective function of exosomes. Overall, the study for the first time indicate a presence of functional ion channel (BK) in exosomes which plays a role in protecting cells and heart against ischemia and reperfusion injury.
Abstract MP221: Early Recruitment Of Neutrophils To The Ischemic Heart Is Orchestrated By Catecholamine-induced Demargination
Authors: Dahdah Albert , Nagareddy Prabhakara R , Sreejit Gopalkrishna , Park Ki Ho H , Jaggers Robert , Johnson Jillian , Ma Jianjie
Published: November 2021

Myocardial infarction (MI) induces a rapid and robust inflammatory response characterized by infiltration of different leukocyte cell types to the infarcted heart. Neutrophils are the first cells to arrive at the infarct where they release a plethora of proteolytic enzymes, exacerbate tissue injury, expand infarct size and promote cardiac dysfunction/ failure. We previously have shown that granulopoiesis (in the bone marrow, BM) is the major source of neutrophils during MI. However, the increase in the number of neutrophils at the infarct begins much earlier than the peak response in the BM (~ 24 hours) suggesting that sources other than granulopoiesis may contribute to the overall neutrophil pool. Because the marginated pool of neutrophils represents the same size of circulating pool, we hypothesized that demargination of neutrophils from the vascular wall contribute to the neutrophil burden in the heart, particularly during the early hours after MI.Using a mouse model of the permanent ligation of the left anterior descending coronary artery, BM ablation of hematopoietic stem cells, flow cytometry and BM transplant techniques, we found that the first wave of neutrophils recruited to the ischemic heart are exclusively sourced from the vasculature and not granulopoiesis in the BM/ spleen. The neutrophils recruited at the heart bore all hallmarks of demargination induced by dexamethasone including decreased F-actin, CD62L, and increased Adam17 expression. The recruitment of neutrophils is orchestrated by catecholamine stress as experimental strategies aimed at blockade of β1 and β2 adrenergic receptors (AR) reduced neutrophil burden in the ischemic heart. Interestingly, despite a decrease in neutrophil burden, the cardiac function did not improve but rather declined. However, short-term inhibition (6-12 hours post-MI) of β AR system either by receptor blockade (propranolol) or inhibition of catecholamine synthesis (AMPT) not only reduced neutrophil burden but also significantly improved cardiac function. Together these data suggest that catecholamine stress induced-demargination constitute the major source of neutrophils during the early hours post-MI. Pharmacological strategies aimed at suppressing the initial onslaught of neutrophils on the ischemic heart may represent a novel and viable approach towards a better resolution of the injury. Currently, studies are underway to define the signaling mechanisms that drive demargination and, to optimize the dose/ duration of a specific β receptor blocker to achieve better functional outcomes.
Abstract MP224: IL-10 Protects The Heart From Late Phage Cardiac Inflammation Associated With Intestinal Permeability Following Transaortic Constriction
Authors: Goswami Sumanta K , Ranjan Prabhat , Verma Suresh
Published: November 2021

Introduction:
Impaired intestinal permeability is known to augment cardiac dysfunction. Though the cardioprotective role of IL-10 is known in early phase cardiac inflammation, following transaortic constriction (TAC), its role in alleviating late phase cardiac inflammation associated with intestinal permeability is not known.

Hypothesis:
We hypothesized that IL-10 reduces late-phase cardiac inflammation by minimizing intestinal permeability and systemic reduction of pathogen-associated molecular pattern (PAMP), which ultimately improve cardiac function.

Methods:
WT and IL-10 KO mice (n=6 in each group) were subjected to TAC or sham surgery, and echocardiography was performed to evaluate cardiac function for up to 56 days. Heart and intestine were collected to evaluate inflammation (RT-qPCR), immune cell infiltration (flow cytometry), and intestinal permeability (WB and immunohistochemistry). Blood was collected to quantify the level of peptidoglycan (PGN, a PAMP) and other bacterial metabolites. The effect of PGN on mitochondrial function was evaluated in neonatal rat ventricular myocyte (NRVM).

Results:
Cardiac functions were significantly reduced in IL10-KO mice, following TAC till 56 days. Furthermore, exacerbated inflammation was observed both at early phase (within 14 days) and late phase (around 56 days) in IL-10 KO TAC mice as compare to the WT counterpart. Interestingly, early cardiac inflammation coincided with immune cell infiltration into the intestine of IL-10 KO mice following TAC. Enhanced inflammatory immune cells homing to the intestine following TAC led to gut dysbiosis (16S RNA sequencing) which ultimately caused impaired gut wall permeability in the IL-10 KO mice as compared to the WT mice. Further, plasma PGN level was significantly increased in IL-10 KO mice following TAC. Finally, our cardiotoxicity array in neonatal rat cardiomyocytes following PGN treatment showed altered expression of proteins which are involved in the regulation of mitochondrial function and cardiac contractility.

Conclusion:
IL-10 plays an important role in limiting late phase cardiac inflammation by reducing intestinal permeability and systemic increase in PGN which induce inflammation and mitochondrial dysfunction in cardiac muscle.
Abstract MP226: Netosis Is Required For S100a8/a9-induced Granulopoiesis After Myocardial Infarction
Authors: Sreejit Gopalkrishna , Jaggers Robert M , Dahdah Albert , Johnson Jillian , Latif Ahmed Abdel , Murphy Andrew , Nagareddy Prabhakara R
Published: November 2021

Myocardial infarction (MI) provokes a massive systemic inflammatory response characterized by enhanced infiltration of neutrophils to the ischemic heart via granulopoiesis in the bone marrow. We recently discovered that S100A8/A9 is released by infiltrating neutrophils in the infarct to induce granulopoiesis by interacting with TLR4 on naïve neutrophils, priming the NLPR3-inflammasome for release of IL-1β which then leads to increased granulopoiesis. However, the mechanism of S100A8/A9 release from the infiltrating-neutrophils remain unclear. We hypothesized that neutrophils release S100A8/A9 via NETosis, a form of cell-death that involves extrusion of decondensed chromatin along with its entire granular content including S100A8/A9. To validate this hypothesis we found that neutrophils sorted from mouse heart 24 hours post-MI were enriched for genetic signature for an Nox-independent NETosis, dominated by Padi4. Furthermore, we found a robust increase in citrullination of histone moieties, colocalization of S100A8/A9 with H3Cit, and increased S100A8/A9 levels in the heart as early as 6 hours after MI. Depletion of Padi4 in the mouse myeloid cells by bone marrow transplantation studies was also associated with suppressed granulopoiesis, fewer neutrophils in the blood and heart, and reduced cardiac and serum S100A8/A9 after MI. To validate these findings in humans, we measured various markers of NETosis and found a marked increase in serum double-stranded DNA, MPO (myeloperoxidase) elastase, which paralleled S100A8/A9 levels in STEMI patients from the time of admission through post-revascularization by percutaneous coronary intervention. Taken together we propose that interventions to reduce NETosis and/or S100A8/A9 release particularly during the acute inflammatory phase could represent a departure from the status quo in managing post-MI inflammation and the subsequent heart failure.
Abstract MP235: PROX1 Contributes To Cardiac Valve Disease
Authors: Ho YenChun , Geng Xin , Varshney Rohan , Kim Jang , Surbrahmanian Sandeep , Alvandi Zahra , Chen Lijuan , Matsuzaki Fumio , Aikawa Elena , Olson Lorin , Ahamed Jasimuddin , Bischoff Joyce E , Srinivasan Sathish
Published: November 2021

Background:
Heart valves regulate the unidirectional forward flow and prevent retrograde backflow of blood during the cardiac cycle. Cardiac valve disease (CVD) is observed in approximately 2.5% of the general population and the incidence increases to ~10% in elderly people. Patients with severe CVD require surgery and effective pharmacological treatments are currently not available. PROX1 is a transcription factor that regulates the development of lymphatic, venous, and lymphovenous valves (vascular valves). We identified that PROX1 is also expressed in a subset of valvular endothelial cells (VECs) that are located on the downstream (fibrosa) side of cardiac valves. Whether PROX1 regulates cardiac valve development and disease is not known.

Method and Results:
We have discovered that mice lacking
Prox1
in their VECs (

Prox1
ΔVEC

) develop enlarged aortic and mitral valves in which the expression of proteoglycans is increased (control, N=10;

Prox1
ΔVEC

, N=9,
p
<0.05). Echocardiography revealed moderate to severe stenosis of aortic valves of

Prox1
ΔVEC

mice (control, N=5;

Prox1
ΔVEC

, N=9,
p
<0.05). PROX1 regulates the expression of the transcription factor FOXC2 in the vascular valves. Similarly, we have found that the expression of FOXC2 is downregulated in the VECs of

Prox1
ΔVEC

mice. Specific knockdown of FOXC2 in VECs results in the thickening of aortic valves (control, N=10;

shFoxc2
ΔVEC

, N=8,
p
<0.05). Furthermore, restoration of FOXC2 expression in VECs (

Foxc2
OE-VEC

) ameliorates the thickening of the aortic valves of

Prox1
ΔVEC

mice (

Prox1
ΔVEC
,

N=9; Foxc2
OE-VEC
;

Prox1
ΔVEC

, N=8,
p
<0.05). We have also determined that the expression of platelet-derived growth factor-B (
Pdgfb
) is increased in the valve tissue of

Prox1
ΔVEC

mice and in PROX1 deficient sheep mitral valve VECs (MVECs) (siCtrl
,
N=4;
siProx1
, N=4,
p
<0.05). Additionally, hyperactivation of PDGF-B signaling in mice results in a phenotype that is similar to

Prox1
ΔVEC

mice (control
,
N=4;

Pdgfb
GOF

, N=3,
p
<0.05).

Conclusion:
Together these data suggest that PROX1 maintains the extracellular matrix composition of cardiac valves by regulating the expressions of FOXC2 and PDGF-B in VECs.
Abstract MP236: PI3Kgamma-PP2A Axis In Regulation Of SERCA Function
Authors: Mohan Maradumane L , Witherow Conner P , Papay Robert , Sun Yu , Stenson Kate , Naga Prasad Sathyamangla V
Published: November 2021

Genetic deletion of Phosphoinositide 3-kinase (PI3Kγ) in mice (PI3Kγ
-/-
) results in increased cAMP levels and enhanced ventricular rate/contractility. We investigated whether PI3Kγ plays a role in cardiac contractility by altering intracellular calcium recycling. Caffeine treatment of adult cardiomyocytes from PI3Kγ
-/-
mice showed significantly reduced calcium reuptake by sarcoendoplasmic reticulum (SR) indicating that PI3Kγ locally regulates SR function. This resulted in elevated levels of intracellular calcium for prolonged period following caffeine. Our findings show that delayed re-uptake of calcium was caused by changes in phosphorylation of phospholamban (PLN), a major regulator of SR calcium reuptake. PI3Kγ
-/-
cardiomyocytes showed significantly reduced PLN phosphorylation due to increase in PLN-associated protein phosphatase (PP) activity as reflected by decreased demethylated-PP2A. Abrogation of PLN phosphorylation in the PI3Kγ
-/-
cardiomyocytes shows that the loss in the steady-state phosphorylation of PLN leads to increased inhibition of SERCA. This inhibition is reflected by the slow reuptake of calcium by the SR in the PI3Kγ
-/-
cardiomyocytes. Concomitantly, significant interaction was observed between SERCA and PLN in the PI3Kγ
-/-
hearts compared to the controls. Consistently, the altered calcium regulation in the cardiomyocytes of PI3Kγ
-/-
can be restored by inhibition of PP by okadaic acid. Unexpectedly, cardiomyocyte-specific overexpression of kinase-dead PI3Kγ (PI3Kγ
inact
) in the global PI3Kγ
-/-
cardiomyocytes normalized caffeine induced calcium reuptake, restored PLN phosphorylation, and decreased PLN-associated PP activity reflected by increased demethylated-PP2A. These studies bring-to-fore an unrecognized kinase-independent regulation of PLN by PI3Kγ through PP2A with implications in deleterious cardiac remodeling as PI3Kγ is significantly upregulated following cardiac stress.
Abstract MP249: Mechanistic Basis And Therapeutic Potential Of Targeting The Non-muscle Rbfox2 Isoform In Myotonic Dystrophy
Authors: Misra Chaitali , Chembazhi Ullas V , Matatov Sarah , Bangru Sushant , Kalsotra Auinash
Published: November 2021

Myotonic Dystrophy type 1 (DM1), the most prevalent form of adult-onset muscular dystrophy, is caused by CTG trinucleotide repeat expansion in the 3’-UTR of the
DMPK
gene. Heart dysfunctions occur in nearly 80% of DM1 patients, and cardiac arrhythmias or conduction abnormalities are a prominent cause of mortality in affected individuals. Yet, the underlying mechanisms causing such abnormalities are not well understood. We recently demonstrated that aberrant expression of a non-muscle splice isoform of RNA-binding protein RBFOX2 triggers cardiac conduction delay, atrioventricular heart blocks, and spontaneous arrhythmogenesis in DM1 hearts. Here we studied the mechanism(s) by which non-muscle RBFOX2 induces mis-splicing of cardiac conduction genes and tested new therapeutic strategies for treating the lethal cardiac symptoms of this disease. By performing eCLIP and high-resolution RNA-sequencing studies on cardiomyocytes isolated from wild type (expressing the normal muscle-specific RBFOX2
43
isoform),
Rbfox2
Δ43/Δ43
(expressing the non-muscle RBFOX2
40
isoform), and RBFOX2
40
overexpressing (OE) mice, we deconstructed the splicing regulatory networks of RBFOX2
43
and RBFOX2
40
isoforms, characterized their respective RNA binding landscapes, and determined the RBFOX2
40
-driven transcriptome alterations in DM1 heart tissue. We acquired induced pluripotent stem cells (iPSC) from healthy, moderate (238 CTG repeats) and severely (1001 CTG repeats) affected DM1 individuals and differentiated them into cardiomyocytes (iPSC-CMs) to generate a human cardiac cell culture model of DM1. Utilizing anti-sense oligonucleotides and RNAi-based approaches, we restored the muscle-specific
Rbfox2
splicing pattern and depleted the non-muscle RBFOX2 isoform in the DM1 IPS-CMs. We are currently analyzing the spontaneous electrical phenotypes of normal and DM1 iPSC-CMs. Collectively, our studies provide an in-depth understanding of the molecular basis for DM1-related electrophysiological abnormalities and offer an avenue to test the potential therapeutic utility of targeting the non-muscle RBFOX2
40
isoform in treating cardiac features of DM1.