bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1105: Inflammatory Bowel Disease Induced Autonomic Dysregulation Increases The Risk For Atrial Fibrillation
Authors: Pelaez Andres F , Varma Disha , Pereira Carlos H , Almeida Jonathas F , Wasserlauf Jeremiah , Keshavarzian Ali , Banach Kathrin
Published: November 2022

Introduction:
Ulcerative colitis causes inflammation of the innermost lining of the colon and rectum. The disease has a relapsing and remitting course and is accompanied by extraintestinal manifestations such as an increased risk for atrial fibrillation (AF). Here we will test the hypothesis that during active colitis, dysregulation of parasympathetic signaling increases the propensity for atrial arrhythmic events.

Methods:
Dextran Sulfate Sodium (DSS: 3%, 7days) treated mice (C57BL/6) exhibit disrupted intestinal barrier function that mimics the disease phenotype of active colitis. Changes in atrial electrophysiology was quantified
in-vivo
(EKG) and in the Langendorff (LD)-perfused heart (atrial electrograms) during peak inflammation (DSS
A
) and remission (DSS
R
).

Results:
During peak inflammation, structural remodeling was reflected in a decreased heart weight to tibia length ratio (Control (CTL
A
): 9.9±0.4 mg/mm, n=10; DSS
A
: 8.8±0.3 mg/mm, n=12; p<0.05) whereas electrophysiological atrial remodeling was supported by a prolonged P-wave duration (CTL
A
: 25.5±0.6 ms, n=10; DSS
A
: 31.2±0.6 ms, n=24; p<0.0001) and shortened atrial effective refractory period (CTL
A
: 27.3±1.7 ms, n=4; DSS
A
: 19.3±0.6 ms, n=4; p<0.01). In comparison to CTL
A
, DSS
A
mice exhibited autonomic dysregulation reflected in an attenuated heart rate (HR) variability and a decreased change in HR in response to the muscarinic receptor blocker atropine (CTL
A
: 7.6±1.8 %, n=5; DSS
A
: 2.2±0.7 %, n=7; p<0.01). The decreased vagal tone in DSS
A
coincided with an exaggerated response to the parasympathomimetic carbachol (CCh, 150ng/g) that resulted in a larger decrease in HR (in vivo: CTL
A
: -12.3±3.1 %, n=10; DSS
A
: -46.0±5.6 %, n=10; p<0.0001) and increased number of spontaneous arrhythmic events (CTL
A
: 1.7±0.6 events/min, n=10; DSS
A
: 9.8±3.8 events/min, n=10; p<0.05) in vivo and in the LD-configuration. The susceptibility for pacing induced AF trended to be increased in DSS
A
animals. Structural and electrophysiological changes were reversible upon remission.

Conclusion:
Active colitis induces transient structural and electrophysiological remodeling. We propose that dysregulation of cholinergic signaling drives the increased propensity for atrial arrhythmia.
Abstract P1107: Lem2 Is Essential For Cardiac Development By Maintaining Nuclear Integrity
Authors: Stroud Matt
Published: November 2022

Background:
integrity is essential for compartmentalisation of nucleus and cytoplasm. Importantly, mutations in -encoding genes are the second-highest cause of familial dilated cardiomyopathy. One such protein that causes cardiomyopathy in humans and affects mouse heart development is Lem2. However, its role in heart remains poorly understood.

Methods:
We generated mice in which Lem2 was specifically ablated either in embryonic cardiomyocytes (Lem2 cKO) or adult cardiomyocytes (Lem2 iCKO) and carried out detailed physiological, tissue and cellular analyses. High resolution episcopic microscopy was used for 3D reconstructions and detailed morphological analyses. RNA-sequencing and immunofluorescence identified altered pathways and cellular phenotypes, and cardiomyocytes were isolated to interrogate nuclear integrity in more detail. In addition, echocardiography provided physiological assessment of Lem2 iCKO adult mice.

Results:
We found that Lem2 was essential for cardiac development, and hearts from Lem2 cKO mice were morphologically and transcriptionally underdeveloped. Lem2 cKO hearts displayed high levels of DNA damage, nuclear rupture, and apoptosis. Crucially, we found that these defects were driven by muscle contraction as they were ameliorated by inhibiting myosin contraction and L-type calcium channels. Conversely, Lem2 ablation in adult cardiomyocytes did not lead to overt cardiac dysfunction up to 18 months of age.

Conclusions:
Our data suggest that Lem2 is critical for integrity at the nascent in fetal hearts, and protects the nucleus from the mechanical forces of muscle contraction. In contrast, the adult heart is not detectably affected by Lem2 loss, perhaps owing to a more established and increased adaptation to mechanical stress. Taken together, these data provide insights into mechanisms underlying cardiomyopathy in patients with mutations in Lem2 and cardio-laminopathies in general.
Abstract P1108: Functional Genomics Analysis Of Sox17 Locus To Define The Pathogenic Role Of FUBP1 In Pulmonary Arterial Hypertension
Authors: Sun Wei , Jiang Danli , Li Gang , Chan Stephen Y
Published: November 2022

Rationale:
Pulmonary arterial hypertension (PAH) is an enigmatic and morbid disease where insights are emerging regarding genetic susceptibility. Genome-wide Association Studies (GWAS) have identified SOX17 as the most significant PAH-associated genomic locus. The allele of tag SNP in this locus is associated with 1.8-fold higher PAH risk. It has been challenging to define the mechanisms underlying the contribution of the PAH-associated functional SNPs (fSNPs) to pathogenesis of the disease.

Methods:
We developed a post-GWAS functional genomics strategy to define the causative fSNPs and identify the associated biological mechanisms. This analysis includes Reel-seq (Regulatory element-sequencing), an EMSA-based high-throughput technique to identify fSNPs in a synthetic DNA library containing PAH-associated SNPs; SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify proteins that specifically bind to fSNPs; and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to show allele-imbalanced binding of these proteins to fSNPs. The regulation of risk gene expression by these fSNP-binding proteins and their pathogenicity were determined in human pulmonary arterial endothelial cells (PAECs) and confirmed in PAH animal models and patients.

Results:
By using high-throughput Reel-seq and subsequent validation with EMSA, intergenic SNP rs4738801 in SOX17 locus was identified as a fSNP from a library containing 254 PAH-associated haplotype SNPs. This fSNP resides in a remote upstream enhancer region of SOX17, an endothelial effector increasingly associated with PAH pathogenesis. Using SDCP-MS and AIDP-Wb, we found that the transcription factor FUBP1 binds to rs4738801 risk allele C with lower affinity than non-risk allele G, resulting in a decrease in SOX17 expression. FUBP1 and target gene SOX17 controlled PAH-associated pathophenotypes in PAECs, including proliferation, apoptosis, and angiogenesis. Downregulated by the major acquired PAH trigger hypoxia, FUBP1 and SOX17 were decreased in lungs and pulmonary ECs isolated from PAH patients and mouse models. A 3.77-fold enrichment of fSNP rs4738801 risk allele C was found in patients with PAH induced by hypoxia, but not in PAH associated with connective tissue disease or congenital heart disease.

Conclusions:
FUBP1 controls SOX17 expression via allele-specific binding to PAH-associated fSNP rs4738801. The reduced binding of FUBP1 to risk allele C defines the genomic architecture contributing to the SOX17-dependent genetic susceptibility of PAH. The downregulation of FUBP1-SOX17 by hypoxia results in endothelial dysfunction, contributing to the acquired pathogenesis of PAH. These findings identify a novel role of FUBP1 in the functional regulation of SOX17 locus and elucidating a pathogenic mechanism that combines the acquired PAH-triggering factors and altered genetic susceptibility.
Abstract P1110: Identification Of Timp-1 As A Downstream Target Of The Coronary Artery Disease Risk Factor Adamts-7 In Atherosclerosis
Authors: Sharifi M. Amin , Wierer Michael , Milic Jelena , Moggio Aldo , Mueller Philipp , Werner Julia , Sager Hendrik , Bernhagen Juergen , Mann Matthias , Schunkert Heribert , Kessler Thorsten
Published: November 2022

Background:
The gene encoding the extracellular matrix (ECM) protease ADAMTS-7 was associated with coronary artery disease (CAD) in genome-wide association studies. ADAMTS-7 is expressed at all stages in human plaques and mice lacking Adamts-7 displayed reduced atherosclerotic plaque formation. While these findings render ADAMTS-7 a promising therapeutic target, the underlying mechanisms remain unknown.

Methods and Results:
Here, we sought to identify downstream mechanisms of ADAMTS-7 in atherosclerotic plaque formation. Targets of Adamts-7 were identified by high-resolution mass spectrometry of atherosclerotic plaques in Apoe-/- and Apoe-/-Adamts7-/- mice. ECM proteins were identified using solubility profiling. The endogenous inhibitor of matrix metalloproteinases (MMP) Timp-1 was identified as a novel target of Adamts-7. Adamts-7 and Timp-1 were found to be co-localized in atherosclerotic plaques and co-immunoprecipitation (Co-IP) studies revealed TIMP-1 as the first putative target to bind to the catalytic domain of ADAMTS-7. In vitro degradation assays demonstrated that ADAMTS-7 degrades TIMP-1. Co-IP furthermore revealed less binding of TIMP-1 to its canonical target MMP-9 when ADAMTS-7 was present. In line, scaffolding and degradation of TIMP-1 by ADAMTS-7 impaired TIMP-1-mediated inhibition of MMP-9 in vitro. As a downstream mechanism, we investigated collagen content in atherosclerotic plaques of Apoe-/- and Apoe-/- Adamts7-/- mice after Western diet. Collagen stainings of the aortic root revealed less collagen as a readout of higher MMP-9 activity in Apoe-/- as compared to Apoe-/- Adamts7-/- mice. Since ADAMTS-7 might exert its pro-atherogenic effects through interaction with TIMP-1, we established a TIMP-1-ADAMTS-7-interaction assay based on Förster resonance energy transfer (FRET) to identify inhibitors of this protein-protein interaction.

Conclusion and Outlook:
TIMP-1 represents a novel downstream target of ADAMTS-7 that can explain the role of this novel risk factor in CAD. Our FRET-based protein-protein interaction assay may be used for high-throughput screening to identifiy inhibitors and prevent the initiation or slow the progression of atherosclerosis.
Abstract P1116: Genetic Mapping Of Cardiomyocyte Ploidy Phenotypes To Identify Genetic Determinants Of Outcomes After Infarction
Authors: Purdy Alexandra L , Kolell Mary E , Knas Makenna , Alvarez Santiago , Foster Parker , Dwinell Melinda R , Saba Laura M , O'Meara Caitlin , Patterson Michaela
Published: November 2022

Outcomes following cardiac injury are influenced by lifestyle and environment, as well as genetic background. Our interest lies in the latter, where very few genes have successfully been linked to heart failure. Contributors to this low success rate include high genetic diversity of patient populations, inability to control for non-genetic factors, and cellular complexities that contribute to outcomes. We propose utilizing the Hybrid Rat Diversity Panel (HRDP) to assess high throughput surrogate phenotypes, which can be predictive of outcomes after injury when measured in the basal state. This approach can offer a solution to the limitations of association studies for complex diseases like heart failure. Our surrogate phenotype of interest is cardiomyocyte ploidy, where high frequency of diploid cardiomyocytes is predictive of ability to regenerate and functional recovery. Conversely, an increased proportion of polyploid (≥8N) cardiomyocytes is associated with adverse ventricular remodeling and dilated cardiomyopathy. Having analyzed 50 of the 96 strains, we see that frequency of the MNDCMs varies from 1.3-20.3% across strains, while frequency of the highly polyploid CMs (≥8N) varies from 0.8-20.9%. Four strains have been selected for myocardial infarction (MI) studies, including M520 (17.4%) and F344 (20.3%) with high MNDCM content and GK (2.8%) and HXB23 (1.8%) with low MNDCM content. Following MI, both M520 and F344 rats exhibit a higher percentage of BrdU+ cardiomyocytes in the border zone region compared to HXB23 rats, supporting the hypothesis that strains with high MNDCM frequency would be better at mounting a proliferative response. This also translated to a greater functional improvement and smaller scars in M520 rats, but not in F344. Additionally, we looked at the size of cardiomyocytes following MI and found that HXB23 rats (20.9% ≥8N CMs) have an increased cross-sectional area in comparison to the other strains, which may be indicative of concentric hypertrophy in response to injury. The HRDP could serve as a powerful tool for mapping traits related to outcomes following MI, allowing us to identify potential gene candidates that may be interrogated further for future use in precision medicine-based therapies. Mapping studies are ongoing.
Abstract P1117: Transcriptional Changes In Failing Hearts In A South Asian Cohort Capture Molecular Pathophysiology Underlying Maladaptive Remodelling And Reflect A Consistent Disease Gene Signature
Authors: Anand Amit , Hellings Samuel , Garg Shilpa , Punnen Julius , Nagamalesh U M , Selvam Sabariya , Raghuraman Bagirath , Date Onkar , Kumaran Senthil , Narayan Pradeep , Udwadia Ankita , Gordon David A , Zhao Lei , Gulia Jyoti
Published: November 2022

Heart failure (HF) is a leading cause of death worldwide and research focused on human cardiac tissue provides insights into underlying disease mechanisms. In this study, we performed transcriptome analyses on cardiac tissue from 21 South Asian (SA) HF patients and compared them with 21 controls without HF. The aim was to i) probe dysregulated genes and pathways in HF and ii) identify a core HF gene signature conserved across etiologies and ethnicities. Gene set enrichment analyses on dysregulated genes (DGs) showed positive enrichment of Gene Ontology terms for ECM remodelling, immune signalling and negative enrichment of terms like cardiac conduction, muscle contraction reflecting altered molecular signalling and cellular cross-talk in failing hearts. The immune cell signatures among the DGs suggest changes in immune milieu which correlates with altered immune signalling pathways. Although poor concordance in DGs across studies on HF transcriptome is well documented, evidence on stratification potential of select DGs is still preliminary. Using an independent component analysis and a machine learning based workflow, we identified gene signatures from two cohorts (SA, GSE116250) independently. We refined these into a core signature after cross validating their stratification potential and by removing the genes with low potential. The core signature effectively separated HF from control samples in SA, GSE116250 and other cohorts potentially reflecting central elements underlying end stage HF. In summary, the transcriptome analyses from SA HF cohort highlights the pathways reflecting HF pathophysiology and reiterates conserved themes across public HF cohorts.
Abstract P1121: Transposition Of Great Arteries - Genetics And Beyond
Authors: Singh Sohrab , Heaton Joseph , Nanavaty Dhairya , Reddy Sarath
Published: November 2022

Background:
Transposition of great arteries (TGA) is one of the most common severe congenital heart defects and neonatal cyanotic heart diseases. Given contribution of several molecular pathways involved in cardiac differentiation (Fig.1), the etiology governing morphogenesis of TGA remains largely unknown. There is greater insight into interactions and molecular variations culminating in aortic-pulmonary transposition. Clinical evidence and genetic sequencing has led to several polymorphic mutations demonstrating phylogenetic conservation of regulatory domains. Defects in transcription factors involved in nodal signaling, in addition to ZIC3 & PROSIT 240, are strongly implicated in arterial transposition. We reviewed current literature on the genetics determining TGA development and associated lateral syndromes

Methods:
We searched PubMed, Scopus, Web of Science, and Google Scholar to identify listed keywords

Body:
TGA is now postulated as part of a spectrum of genetic disorders with familial recurrence, rather than sporadic defect. Evidence exists for TGA's correlation with genes involving lateralization defects, heterotaxy and asplenia. Evidence, albeit scarce, has surfaced depicting association of TGA with genetic syndromes like Trisomy 8, 18, VACTERL and CHARGE syndrome, Tuberous sclerosis, Deletion 11q, 18p, and other genetic syndromes like Digeorge, Turner’s, Noonan’s, Williams, and Marfan. Additional gene mutations causing TGA might include CFC-1, TRAP2, FOXH1, LEFTY2, PITX2, GDF1. The interrelation between mutations in ciliary genes contributing to both TGA and neurodevelopment disorder, prompts consideration of common ciliopathic pathogenies explaining association between TGA and neurodevelopmental disorders.

Conclusion:
The occurrence of mutations in a single gene, though a cause for TGA, does not correlate with 5-7% occurrence rate for TGA. Major research has focused on a single gene approach, neglecting the simultaneous screening for other susceptible targets. The multiple-hit model suggested by De Luca et al. (2010) is a promising approach for further understanding the contribution of multi-gene sequences. Minor genetic variations in several genes may have a cumulative effect that increases disease susceptibility in TGA and other congenital heart defects
Abstract P1122: Coupled Myovascular Expansion Directs Cardiac Growth And Regeneration
Authors: DeBenedittis Paige , Karpurapu Anish , Henry Albert , Thomas Michael C , Lumbers Tom , Karra Ravi -
Published: November 2022

Background:
Heart regeneration requires multiple cell types to enable cardiomyocyte (CM) proliferation. How these cells interact to create growth niches is unclear. Here we profile proliferation kinetics of cardiac endothelial cells (CECs) and CMs in the neonatal mouse heart and find that they are spatiotemporally coupled.

Methods and Results:
We show that coupled myovascular expansion during cardiac growth or regeneration is dependent upon VEGF-VEGFR2 signaling, as genetic deletion of Vegfr2 from CECs or inhibition of VEGFA abrogates both CEC and CM proliferation. Repair of cryoinjury displays poor spatial coupling of CEC and CM proliferation. Boosting CEC density after cryoinjury with virus encoding Vegfa enhances regeneration. Using Mendelian randomization, we demonstrate that circulating VEGFA levels are positively linked with human myocardial mass, suggesting that Vegfa can stimulate human cardiac growth.

Conclusions:
Our work demonstrates the importance of coupled CEC and CM expansion and reveals a myovascular niche that may be therapeutically targeted for heart regeneration.
Abstract P1126: Development Of A Tissue Engineered Small Vascular Graft With Human Amnion Membrane
Authors: Wang Bo , Wang Xiaolong
Published: November 2022

The vascular transplantation is an established therapy for vascular diseases by replacing or bypassing the damaged blood vessel with a functional vascular graft. Autologous blood vessels are considered as the gold standard grafts for vascular transplantation, but the limited tissue source and the high morbidity associated with tissue harvesting have restricted their therapeutic potential. Vascular tissue engineering represents an alternative solution to replace the autologous blood vessels by developing an artificial conduit which can closely mimic the nature blood vessel. However, clinical
trials with
small vascular grafts are
disappointing.
The aim of this study is to investigate the feasibility of constructing a small vascular graft from the decellularized amniotic membrane (DAM graft) via different crosslinking methods. The human amnion membrane was decellularized, trimmed into 5 cm x 8 cm rectangular shape, and rolled around a catheter (diameter =1.2 mm) to make the primary DAM graft. Each of the resulting conduits was additionally crosslinked with: a) 0.1% glutaraldehyde; b) 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (20mM) / N-hydroxy succinimide (10mM) (EDC-NHS); c) 0.5% genipin; and d) no-crosslinking as control group. Our results demonstrated the structural integrity, material stability, mechanical behaviors, and biocompatibilities of the DAM grafts can be modified by the chemical crosslinking treatment. In which, the genipin crosslinked DAM graft exhibited the most integrated layered structure. Its level of material stability was comparable to those of the glutaraldehyde and the EDC-NHS crosslinked DAM grafts. Furthermore, the genipin crosslinking did not significantly affect the mechanical properties of the DAM graft. The genipin-crosslinked DAM graft was further tested
in vivo
by implanting it in a rat abdominal aorta replacement-model. All rats implanted with the genipin-crosslinked DAM graft exhibited a blood vessel patent up to 9-month post-implantation. In conclusion, we represented a
potential
avenue to construct
a
flexible vascular graft that has the patient-specific dimension and meet specific demands for biological and mechanical performance criteria by using the DAM followed by lyophilization and the genipin.
Abstract P1131: Modeling Contractile Diseases Using Scalable 3D Engineered Heart Tissues For Drug Discovery
Authors: Geisse Nicholas , Berry Bonnie , Gray Kevin , Kharoufeh Samir , Luttrell Shawn M , Macadangdang Jesse , Worthen Christal
Published: November 2022

Model systems that accurately recapitulate healthy and diseased function in a dish are critical for the development of novel therapeutics. For cardiac diseases, direct assessment of contractile output constitutes the most reliable metric with which to assess overall tissue function, as other ‘proxy’ measurements are poor predictors of muscle strength. 3D engineered muscle tissues (EMTs) derived from iPSCs hold great potential for modeling contractile function. Here, we have developed a platform and device that utilizes 3D EMTs in conjunction with a label-free magnetic sensing array. The platform enables facile and reproducible fabrication of 3D EMTs using virtually any cell source and is coupled with a highly parallel direct measurement of contractile strength. This approach enables the stratification of healthy and diseased phenotypes and facilitates compound safety and efficacy screening for evaluation of a drug’s effect on contractile output. We will present data from a drug (BMS-986094) that failed clinical trials due to unanticipated cardiotoxicity. We go on to show both the acute and chronic effects of doxorubicin in cardiac EMTs. Contractile force decreased in a dose dependent-like manner when the drug was applied continuously. Interestingly, a single 1uM bolus induced a transient effect that could be washed out over time. A repeat bolus, however, irreversibly abolished contraction, suggesting that repeat dosing may have a cardiotoxic effect on the muscle. These data demonstrate a first-and-only commercial platform for high-throughput assessment of 3D cardiac muscle contraction with potential for widespread adoption within the drug development field.
Abstract P1135: Psme4 Degrades Acetylated Yap1 In The Nucleus Of Mesenchymal Stem Cells To Induce Cardiac Commitment
Authors: Yoon Somy , Kim Yong S , Eom Gwang Hyeon , Ahn Young Keun
Published: November 2022

Intensive research has focused on minimizing the infarct area and stimulating endogenous regeneration after myocardial infarction. Our group elucidated that apicidin, a histone deacetylase (HDAC) inhibitor, robustly stimulates cardiac commitment of mesenchymal stem cells (MSCs) through acute loss of YAP1. Here we further studied the mechanism of this role of YAP1 in MSCs. We found that acute loss of YAP1 after apicidin treatment resulted in the mixed effects of transcriptional arrest and proteosomal degradation. Subcellular fractionation revealed that YAP1 was primarily localized in the cytoplasm. YAP1 was acutely relocalized into the nucleus and underwent proteosomal degradation. Interestingly, phosphor-S127 YAP1 was shuttled into the nucleus, suggesting that a mechanism other than phosphorylation governed subcellular localization of YAP1. Apicidin successfully induced acetylation and subsequent dissociation of YAP1 from 14-3-3, an essential molecule for cytoplasmic restriction. HDAC6 regulated both acetylation and subcellular localization of YAP1. An acetylation-dead mutant of YAP1 retarded nuclear redistribution upon apicidin treatment. We failed to acquire convincing evidence for polyubiquitination-dependent degradation of YAP1, suggesting that a polyubiquitination-independent regulator determined YAP1 fate. Nuclear PSME4, a subunit of the 26S proteasome, recognized and degraded acetyl YAP1 in the nucleus. MSCs from PSME4-null mice were injected into infarcted heart and aberrant sudden death was observed. Injection of immortalized human MSCs after knocking down PSME4 failed to improve either cardiac function or the fibrotic scar area. Our data suggest that acute ablation of YAP1 in the nucleus by the acetylation-dependent proteasome subunit PSME4 is mandatory for cardiac commitment of MSCs.
Abstract P1136: Amino Acid Availability Regulates Differentiation Of HIPSC-derived Cardiomyocytes In A Protein-free System
Authors: Lyra Leite Davi M , Copley Raymond R , Freeman Phillip P , McKenna Donald E , Fetterman Katherine A , Shah Disheet A , Negahi Shirazi Ali , Fonoudi Hananeh , Mohamed Tarek M , Blancard Malorie , Burridge Paul W
Published: November 2022

Traditional protocols for the differentiation of hiPSC-derived cardiomyocytes (hiPSC-CMs) rely on complex media and the use of animal or recombinant proteins either directly or in supplements, such as B27, to obtain tissue monolayers. This fact decreases their reproducibility, while potentially increasing their costs and limiting higher-throughput production of cardiomyocytes for applications in both regenerative medicine, disease modeling, and pharmacological screenings. Albumin/serum-free protocols have been developed but produce cells with limited functionality and at lower yields, despite them being TNNT2-positive. We previously demonstrated it is possible to simplify the differentiation protocol utilizing a stepwise approach and combining time-point interventions with small molecules or nutritional inputs. Here, we performed a thorough screening of commercial media and their suitability for differentiation of hiPSC-CMs. We identified suitable basal media formulations and developed a novel protein-free differentiation protocol (PFDM) for generating hiPSC-CMs that preserves purity, yield, morphology, and functionality while reducing the cost of the differentiation process. PFDM also enabled the identification of nutritional requirements essential for cardiac differentiation and allowed a detailed investigation into how some common components supplemented during hiPSC-CM differentiation impact tissue morphology and contractility. We showed that non-essential amino acids regulate differentiation in a time-dependent manner. Similarly, antioxidant agents such as sodium pyruvate and ascorbic acid, while essential for differentiation, are potentially harmful during early cardiac mesoderm induction. Lastly, we validated this PFDM methodology in 35 control and patient-derived hiPSC lines, with monolayer differentiations in 6- and 12-well plates, 15 cm dishes, in addition to differentiations in 3D using 100- and 250-mL spinner flasks.
Abstract P1141: Heparinized Alginate And Collagen-based Hydrogels Enhance Localized Vascularization In Ischemic Tissue
Authors: Roser Stephanie M , Minor Alicia , Polucha Collin , Coulombe Kareen L
Published: November 2022

Over 800,000 myocardial infarctions (MI) occur annually in the United States alone, 25% of which represent a repeat occurrence further indicating chronic ischemia in the myocardium. Cardiovascular regeneration is largely dependent on the stimulation of blood vessel development for recovering tissue perfusion and modulating ventricular remodeling. The delivery of angiogenic growth factors and cytokines has demonstrated the ability to initiate angiogenesis and vascular network formation in cardiac tissue, with localization of bioactive molecules more effective than systemic delivery. Alginate, with its high biocompatibility and biodegradation, represents a promising hydrogel material for factor delivery due to its high protein loading capacity. We hypothesize that covalently binding heparin to alginate will increase binding and retention to subsequently lengthen the local release duration of vascular-promoting factors. For greater tuning and biointegration, collagen and alginate hydrogel designs were optimized for both injection and co-implantation of a solid film with hiPSC-CM based ECTs in a rat model of myocardial infarction. Injectable and solid formulations showed stiffnesses of 50 +/- 2 kPa and 900 +/- 193 kPa, respectively, which was maintained for up to 6 hours of dry time. Release profiles showed sustained growth factor release, with 65% of the total content released at 7 days and 100% release in 14 days.
In vitro
evaluation using human induced pluripotent stem cell-derived endothelial cells (hiPSC-ECs) showed cell viability and primitive network formation.
In vivo
subcutaneous injections showed host-vessel penetration into the delivered biomaterial within 4 days. Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) loaded injections demonstrated an average lumen density of 80 +/- 15 vessels/mm
2
when compared to 4 +/- 1 vessels/mm
2
in the unloaded controls. In conclusion, we have designed a biomaterial system for disparate delivery models and shown successful vascularization of the biomaterial, demonstrating the versatile vascular regenerative capabilities of the hydrogel design.
Abstract P1145: Hydrogel Matrix Support Of Suspension-differentiated Hipscs Impacts Resulting
In Vitro
Cardiac Tissue Functionality
Authors: Hashemi Mohammadjafar , Finklea Ferdous , Hammons Hanna , Tian Yuan , Wright Katherine , Lipke Elizabeth A
Published: November 2022

Functional in vitro cardiac tissues are needed to provide insight into the cellular level of disease. Previously, we demonstrated hydrogel-encapsulated hiPSCs undergo cardiac differentiation and achieve cardiomyocyte (CM) maturation including T-tubule formation. To investigate the effect of encapsulation on differentiation and cardiac tissue functionality, here we compared PEG-fibrinogen (PF) microsphere (MS)-supported differentiation approach to self-aggregated embryoid body (EB) differentiation. Briefly, hiPSCs were encapsulated or formed into EBs; cardiac differentiation was initiated by Wnt activation and subsequent inhibition. CM content, contractile functionality, drug response, and electrophysiological properties were determined by flow cytometry, microscopy, and optical mapping, respectively.Initial diameter was 673±22 μm (coefficient of variance, COV=0.03) for MS and145±34 μm (COV=0.24) for EBs. The number of CMs produced (differentiation day 10) per initial hiPSC was significantly higher for MS (2.8 ± 1.8) than EBs (0.6±0.12). MS also yielded a higher percentage of CMs; MS-encapsulated hiPSC differentiation resulted in 74.7±13.8% CMs while for EBs it was 56±16.1 % (cTnT+, MF20+, n=14 and 5 batches, respectively, p<0.05). Myocardial contractility was also enhanced in cardiac MS as compared to EBs (similar CM content, day 10); maximum contraction velocity was four times higher for MS than EBs (139±17.5 μm/s versus 33.4±5.1 μm/s) and maximum relaxation velocity was fourteen times higher (97±7.2 μm/s versus 7.4±0.4 μm/s).Furthermore, cardiac MS responded more vigorously to isoproterenol (IP) and propranolol (P) (frequency +124% (IP), - 61% (P), versus +49% (IP) and -27%(P) for EB), suggesting that MS-CM β-adrenergic signaling may be more mature. Calcium transient propagation was more uniform in MS-CMs compared to EB-CMs; calcium transient duration (CTD) at 50% and 80% repolarization and calcium transient velocity were similar. In conclusion, MS supported cardiac differentiation supported higher CM content, faster contractility, and improved drug response in comparison to similarly differentiated EBs, demonstrating the potential of encapsulation to enhance cardiac tissue functionality for disease modeling.
Abstract P2005: Loss Of Full-length Mylk3 Causes Dilated Cardiomyopathy Via A MYL2-independent Mechanism
Authors: Williams Jack L , Paudyal Anju , Stewart Michelle , Cutillas Pedro , Tinker Andrew , Metherell Lou
Published: November 2022

Introduction:
We recently discovered C57BL/6N mice develop dilated cardiomyopathy (DCM), while the closely related C57BL/6J do not. We identified a variant (Chr8:85365179A>T, c.-5T>A) in myosin light chain kinase 3 (
Mylk3
) in the C57BL/6N substrain that abolishes translation of the predominant transcript (ENSMUST00000034133). MYLK3 phosphorylates many sarcomere proteins, in particular myosin light chain 2 (MYL2), vital for cardiac development and function.

Hypothesis:
The MYLK3 variant is sufficient to drive the development of DCM.

Results:
We generated a novel isogenic mouse line carrying the
Mylk3
c.-5T>A variant on the C57BL/6J background (C57BL/6J
Mylk3.6N
). We found symptoms of DCM in male mutant mice at just 4 weeks old, with increased left ventricular diameter and volume, thinning of the posterior wall and a reduced ejection fraction. These data suggest the
Mylk3
variant alone is sufficient to cause DCM. Surprisingly, we found no difference in phosphorylation of myosin light chain 2 (MYL2) by Phos-Tag® blotting at any age. This was unexpected, as previous reports in
Mylk3

-/-

mice found complete loss of MYL2 phosphorylation with a similar profile of DCM. However, in our mouse model, loss of MYLK3 is caused by a single point mutation in exon 1 which ablates the primary isoform (ENSMUST00000034133; Q3UIZ8) but western blotting with a C-terminal antibody revealed expression of a shorter isoform that is unaffected by the variant due to a distinct N-terminus (ENSMUST00000122452; D3Z630). There was no difference between the isoforms in their ability to phosphorylate MYL2 suggesting the shorter isoform can compensate. MYLK3 may also interact with ACTN2 and phosphorylate TNNI3. We found no difference in the expression or localisation of ACTN2 in our tissues, although co-IP in cells showed the primary isoform binds more readily to ACTN2, however, there was a significant reduction in TNNI3 S23/24 phosphorylation at all ages in C57BL/6J
Mylk3.6N
mice. This could affect cross-bridge cycling kinetics by altering the function of TNNI3 within the tropomyosin/troponin complex.

Conclusions:
Loss of full-length MYLK3 causes DCM via a MYL2-independent mechanism, possibly due to lack of phosphorylation of alternative target(s), such as TNNI3.
Abstract P2006: S100A1 And STRIT1 Redundantly Governs Responsiveness Of The Heart To Hemodynamic Stress Via Modulation Of SERCA2a Activity
Authors: Jebessa Zegeye Hailu , Schneider Andrea , Dewenter Matthias , Berlin Michael , Makarewich Catherine A , Olson Eric N , Freichel Marc , Busch Martin , Backs Johannes , Most Patrick
Published: November 2022

Background & Objectives:
SR Ca2+ load, which is sustained by SERCA2a pump activity, is a critical determinant for cardiac performance regulation & adaptation. Independent studies identified both S100A1 & STRIT1 as molecular enhancers of SERCA2a activity in the heart. S100A1 & STRIT1 decline in post-myocardial infarction hearts aggravated the transition to adverse cardiac remodelling & contractile failure. We therefore hypothesized that S100A1 & STRIT1 could act as independent but potentially redundant molecular switches for SERCA2a activity.

Methods & Results:
S100a1 knock-out (SKO) mice display no overt cardiac contractile or structural abnormalities in the absence of stress. RNA-seq transcriptomic analysis of left ventricle (LV) of SKO & wild type (WT) identified Strit1 amongst the top 3 most upregulated genes in SKO LV. We validated STRIT1 upregulation by RT-PCR as well as by immunoblotting (IB) that yielded a 15-fold increase compared with WT. Age lapse-resolved RT-PCR analysis showed Strit1 response to S100a1 knockout begins at post-partum day 5 & reaches plateau in adulthood. Next, we generated Strit1-S100a1 double knock out mice (StSKO), which showed only a mild decline in cardiac contractile performance. Interestingly, LV tissue serin-16 phospholamban (PLN) phosphorylation levels & PLNs pentameric state were found to be enhanced. WT, SKO & StSKO mice were then subjected to transaortic constriction (TAC) & followed for 60 days, which fully unmasked the mutually compensatory functions of S100A1 & STRIT1. In TAC-StSKO hearts showed significantly higher decline in LV %EF, significantly increased LV end-systolic volume & LV end-systolic diameter, & significantly increased cardiac hypertrophic growth together with concordant molecular markers. TAC-SKO mice did not show any decline in STRIT1 protein upregulation, while TAC-WT hearts showed a putatively compensatory increase in the S100A1/STRIT1 protein ratio.

Conclusion:
Our first results indicates that STRIT1 & S100A1 can act as compensatory molecular switches securing sufficient SERCA2a activity. As such, our study further sheds new light onto the novel concept of “molecular redundancy” to secure & protect cardiac key effector activities to cope with distinct hemodynamic stressors
Abstract P2012: GRK2-Induced Cardiomyocyte AdipoR1 Phosphorylation Blocks Adipocytes-Derived Exosomes Cardioprotection Against Ischemic Heart Failure
Authors: Zhang Zhen , Zhu Di , Meng Zhijun , Zhao Jianli , Zhao Fujie , Yao Peng , tsukuda jumpei , Liu Demin , Gan Lu , Lau Wayne Bond , Xie Dina , Lopez Bernard L , Christopher Theodore , Gao Erhe , Koch Walter , Ma Xinliang , Wang Yajing
Published: November 2022

Background:
Despite significant reduction in acute MI death, ischemic heart failure (IHF) and resultant death continually escalate with incompletely understood mechanism. We recently reported that adipocyte-derived exosomes (ADp-Exo) protect heart from acute MI/R injury. However, it remains unknown whether and how cardiomyocytes (CM) response to ADp-Exo is altered during the chronic phase of MI, negatively impacting IHF development.

Methods and Results:
Intramyocardial injection of ADp-Exo (isolated from epididymal fat pad) immediately after MI (90 min MI/4 weeks reperfusion) significantly attenuated post-MI remodeling. However, the protective effects were completely lost when ADp-Exo were administered 1 week after MI. To identify the molecular mechanisms responsible for ADp-Exo cardioprotection and its alteration during chronic MI, a series of in vitro experiments were performed. Adiponectin (APN), a potent cardioprotective adipokine, was detected on the surface of ADp-Exo (Exo-flow kit). Treatment of CM with ADp-Exo activated multiple cell salvage kinases (e.g., ACC, ERK and AMPK). These effects were lost in APN neutralization antibody pre-incubated ADp-Exo or ADp-Exo from APNKO mice, suggesting Exo surface APN may mediate ADp-Exo cardioprotection. Moreover, ADp-Exo cell salvage kinase activation effect was absent in CM from AdipoR1KO mice or GRK2 transfected CM, suggesting GRK2-induced AdipoR1 phosphorylation at Ser
205
(as we demonstrated in Circulation, 2015 and Circ Res, 2022) is likely responsible for incapability of ADp-Exo protection during post-MI remodeling. To obtain direct evidence supporting this novel hypothesis, AdipoR1
S205A
or AdipoR1
S205E
(pseudo-phosphorylation) mice were generated. ADp-Exo cardioprotection was restored in AdipoR1
S205A
mice, even when they were administered 1 week after MI. In contrast, ADp-Exo cardioprotection was lost in AdipoR1
S205E
mice, even when they were administered immediately after MI.

Conclusions:
We demonstrate for the first time that, APN located on the surface of ADp-Exo acts as the critical executor of ADp-CM communication, mediating ADp-Exo cardioprotection. GRK2-induced CM AdipoR1 phosphorylation blocks ADp-Exo protective action, contributing to IHF progression.
Abstract P2015: Metabolic-epigenetic Coupling In High Fat Diet And Heart Failure With Reduced Ejection Fraction
Authors: Gromova Tatiana , Kimball Todd H , Ren Shuxun , Wang Yibin , Chapski Douglas J , Vondriska Thomas M
Published: November 2022

Given rising obesity rates, understanding how metabolic changes exacerbate other cardiovascular disease (CVD) risk factors will yield novel therapeutic targets for associated comorbidities, such as heart failure. We hypothesize that obesity-related metabolic syndrome induces transcriptional programs that engender cardiac pathology via mechanisms involving chromatin regulators, thereby perpetuating the pathology by epigenetic memory. C57BL/6J adult male mice were put on a high fat diet (HFD) for 24 weeks. After HFD, hearts were in hypertrophy, as defined by an increase in heart weight to tibia length ratio (24.67 ± 5.14 mg/mm in HFD versus 17.06 ± 0.98 mg/mm in regular chow controls, p=0.026). Examination of blood plasma revealed significant increase in LDL-C (74.37 ± 22.45 mg/dL in HFD versus 21.91 ± 5.98 mg/dL in controls, p=0.0005) and insulin (452.81 ± 224.77 uIU/mL in HFD versus 89.66 ± 45.60 uIU/mL in controls, p=0.0003), indicative of metabolic disorder. RNA-seq of isolated cardiomyocytes revealed distinct transcriptomic changes at 1 week, 6 weeks, and 24 weeks of HFD. At 1 week, HFD stimulus induced promotion of lipid metabolism (
Pdk4, Per2
) and suppression of genes related to development and proliferation (
Sox4, Epha4
). By 6 weeks, the cardiomyocytes underwent metabolic reprogramming (downregulation of
Lpl
and
Irs2
) and structural changes (
Acta2, Col8a1).
At 24 weeks, we observed suppression of ketolytic machinery (downregulation of
Abat, Bdh1,
and
Oxct1
) in response to obesity-driven hyperglycemia, suggesting that cardiomyocytes transitioned to a pre-diabetic state. To test whether this metabolic disease state compounds with other CVD risk factors, we devised a two-hit model, where mice were fed HFD for 6 weeks and then underwent transverse aortic constriction as a model of pressure overload. Cardiomyocytes from this two-hit model exhibited an enhanced hypertrophic response (
Myh7, Nppa, Col1a1
mRNA levels) compared to pressure overload with regular chow, sham surgery with HFD, or sham surgery with chow. Thus, phenotypic and transcriptional data suggest that HFD reprograms cardiomyocytes to a pre-diabetic state characterized by insulin resistance which exacerbates the effects of other CVD risk factors like hypertrophy.
Abstract P2016: Gut Microbiota-derived Short-chain Fatty Acids Benefit Cardiac Extracellular Matrix Remodeling In Pressure Overload
Authors: Lin Chen-Ju , Cheng Yu-Che , Chen Hung-Chih , Chao Yu-Kai , Nicholson Martin , Yen Eric , Kamp Timothy , Hsieh Patrick C
Published: November 2022

Introduction:
The gut microbiota has been implicated in various heart diseases by producing metabolites that modulate host immunity and metabolism. In our previous study, we found that mice with dysbiosis suffered more from cardiac rupture, resulting in higher mortality than mice possessing commensal gut flora after myocardial infarction. This suggests that there are unexplored routes to cardiovascular health through the gut microbiota.

Hypothesis:
We hypothesize that a community of symbiotic gut microbiota is required for heart mechanical modifications during adaptive cardiac remodeling under stress.

Methods and Results:
Pressure-overload stress was induced by a transverse aortic constriction (TAC) surgery and dysbiosis was induced by antibiotic treatment (ABX) in mice. Under echocardiography, ABX-TAC mice showed worse cardiac outcomes versus controls (ejection fraction = 59% ± 2% vs 67% ± 2%, P < 0.001, N ≥ 12/group). Under microscopic examination of the extracellular matrix and tensile tests, ABX-TAC mice had larger fibrotic areas (9.4% ± 0.4% vs 5.7% ± 0.4%, P < 0.0001) and collagen disarray, accompanied by more severe ventricular stiffening (Young’s moduli = 360 ± 10 kPa vs 280 ± 43 kPa, P < 0.01, N ≥ 6/group). When establishing normal gut flora before surgery, germ-free mice had heart malfunctioning rescued (change of myocardial performance index = 15% ± 13% vs 120% ± 32% untreated, P < 0.05, N ≥ 5/group). The normal gut flora was profiled by third-generation 16S sequencing to acquire bacterial information with high accuracy, followed by PICRUSt analysis revealing that the microbes favored the production of short-chain fatty acids (SCFAs) (N ≥ 8/group). When supplemented with SCFA before surgery, ABX mice gained better cardiac outcomes (ejection fraction = 65% ± 2% vs 53% ± 3% untreated, P < 0.01, N ≥ 5/group). Cardiac fibroblasts treated with SCFA were less susceptible to TGF-β1-triggered fibrogenesis (COL1A1/GAPDH = 0.8 ± 0.3 vs 2.3 ± 0.3 untreated, P < 0.01, n ≥ 4/group).

Conclusions:
In conclusion, our study demonstrates firstly that gut microbiota-derived SCFAs manipulate heart mechanical functioning under stress potentially acting via cardiac fibroblasts. This provides new insights into the management of heart diseases through the gut microbiota.
Abstract P2020: A Novel Molecular Mechanism Of Cardiac Hypertrophy
Authors: Lin Yongluan , Lu Lei , Zheng Liling , Zhong Shuping
Published: November 2022

Proliferation of cardiac myocytes is accompanied by increases in cell mass to maintain myocyte size in fetal heart. In adult cardiac myocytes, cell growth becomes uncoupled from proliferation after growth stimuli, resulting in hypertrophic growth. Both proliferation and hypertrophy growth of cardiac myocytes require more protein synthesis. Brf1 (TF IIB-related factor 1) and Pol III genes (RNA Polymerase III-dependent genes) are the dominant factors of protein produce. Brf1 is a key regulator for Pol III genes. Cardiac hypertrophy is a critical risk factor for cardiovascular disease, including heart failure, arrhythmia, and sudden death. Cardiac hypertrophy involves both the processes of embryonic gene expression and transcriptional reprogramming. The processes are tightly modulated by different signal pathways and epigenetic regulation. Diabetic cardiomyopathy is associated with an increased risk for heart failure and death in patients with diabetes. To explore the molecular mechanism of cardiac hypertrophy, we investigate the alterations of Brf1 and Pol III gene transcription under glucose treatment by using cell culture model and animal model. We have found that glucose increases Brf1 expression and Pol III gene transcription. We identified that these genes are mediated by JNK1, MSK1, pAMPK and phosphorylated histone H3. inhibiting the pathways decrease Brf1 expression, reduce Pol III gene transcription, consequently repression of myocytes growth. It implies that developing an effective inhibitor to repress Brf1 expression may provide a potential approach for the therapy of cardiac hypertrophy. More studies on cardiac hypertrophy are going in our laboratory.