bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 297: Effects of p53 Stabilization During Heart Failure Progression on Telomere Repeat Binding Factor 2 and Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α in the SHHF Rat
Authors: Nelson Christopher B , Chicco Adam J , Bailey Susan M 
Published: July 2021

The tumor suppressor protein p53 is a cellular stress sensor and transcription factor classically accepted to target the transcription of genes that induce senescence and apoptosis. Diverse stressors including genomic or telomeric DNA damage, telomere uncapping, hypoxia, oxidative stress, and mechanical stress increase stable cellular p53. In models of heart failure and in human failing hearts, p53 is increased. High p53 has also been suggested to be necessary for the transition from hypertrophy to heart failure.
In recent years it has become clear that senescence and apoptosis may not be the only significant deleterious effects of increased cellular p53. It has been suggested that p53 can decrease mitobiogenesis and cellular ATP production by direct repression of PGC1-a expression. Additionally, P53 has been shown to stimulate the expression of Siah1, an E3 ubiquitin ligase that mediates degradation of the telomere binding protein TRF2. TRF2 loss is a form of telomere uncapping that, in cell culture models, causes a subsequent DNA damage response including further p53 stabilization. The relative importance of these p53 linked pathways has yet to be explored in the progression of hypertensive heart failure.
Spontaneously Hypertensive Heart Failure rats (SHHF) rats were sacked at four stages during heart failure progression and LV removed for western blot analysis. Compared to young (2-4 month) rats, 14-15 month rats showed significantly increased p53 levels (2.3 fold), normal TRF2 levels, and slightly decreased PGC1-a (.98 fold). Old SHHFs (20-22mo) had p53, TRF2, and PGC1-a levels similar to 14-15 month rats (2.1, 1.09, and.83 fold, respectively). Rats with end stage heart failure showed a significant 4.5 fold increase in p53 expression, with a corresponding decrease in TRF2 (.74 fold, p=.087), and increase in PGC1-a (1.3 fold, nonsignificant).
These data are in agreement with previous suggestions that increased p53 is most important during the transition from hypertrophy to heart failure, and suggest that levels of p53 encountered in the SHHF model have little effect on PGC1-a and mitobiogenesis, but may mediate an increase in telomere uncapping during heart failure.
Abstract 313: Changes in Autophagic Flux in Patients Undergoing Cardiac Surgery
Authors: Jahania Salik , Sengstock David , Vaitkevicius Peter , Mahmood Redah , Sabagh Fahed , Hagood Kristyn , Ito Bruce , Andres Allen , Gottlieb Roberta A , Mentzer Robert M 
Published: July 2021

Autophagy is an endogenous survival mechanism and adaptive response to cellular stress. We hypothesized that autophagy plays an important cardioprotective role in humans and would be accelerated during the ischemic stress of conventional heart surgery. In a prospective study to test this hypothesis, we measured key autophagy proteins (Beclin-1, Atg5-12, p62) in cardiac tissue from 5 patients undergoing cardiac surgery. Right atrial biopsies were obtained before and after cessation of cardiopulmonary bypass (CPB). In 4 of these patients, CPB was associated with a marked decrease in cardiac Beclin-1, Atg5-12, and p62. We believe that depletion of these factors, particularly p62, reflects a brisk increase in autophagic flux (Fig. 1). In one patient (#02), autophagic flux appeared to be impaired. This occurred in an aged patient with characteristics of metabolic syndrome (MetS) who had the highest predicted operative morbidity/mortality. While the number of patients studied is small and insufficient to reach any definitive conclusions, these preliminary data confirm the feasibility of studying autophagy in the human heart and suggest that ischemic stress during heart surgery is associated with a marked upregulation of autophagic flux. A better understanding of the role of autophagy could lead to the development of new cardioprotective strategies.
[Figure 1: Changes in cardiac autophagy proteins in patients undergoing heart surgery before and after cross-clamping the aorta (after restoration of myocardial blood flow).]
Abstract 327: Utrophin Loss in the Absence of Dystrophin Induces Alterations in Cardiac-Related Protein Expression and Activity in the Heart and Muscle of Dystrophic Mice
Authors: Schneider Joel S , Ramachandran Jayalakshmi , Shanmugam Mayilvahanan , Crassous Pierre-Antoine , Gonzalez James P , Gordan Richard , Xie Lai-Hua , Beuve Annie V , Babu Gopal J , Peluffo Ruben D , Fraidenraich Diego 
Published: July 2021

Duchenne Muscular Dystrophy (DMD) is an x-linked recessive disorder that affects 1:3500 males. The disease is characterized by the absence of the dystrophin protein, leading to progressive muscular degeneration in both skeletal and cardiac muscle. DMD is recapitulated in mouse models, the most severe model lacking both dystrophin and its compensatory homolog, utrophin (mdx:utro). This model mimics the onset of DMD much more rapidly than mdx. Nitric oxide (NO) synthesis is significantly diminished in DMD and mdx skeletal muscle. We have identified previously unreported functional differences in neuronal nitric oxide synthase (NOS1)-related nitric oxide production in cardiac musculature in mdx:utro mice, caused by the absence of utrophin. We have quantified mechanistic differences in L-Arginine mediated nitric oxide production via NOS1 in mdx and mdx:utro myocytes isolated via Langendorff perfusion. Interestingly, L-arginine transport levels were increased in the presence of this amino acid and occurred concurrently with increases in the number of mRNA transcripts for the low-affinity cationic amino acid transporter, CAT-2A. As CAT activity is an obligate process for L-Arginine entry to the cardiac myocytes, this may represent a potential compensatory mechanism related to decreased nitric oxide production.
We have also found manifestations of ectopic cardiac-related protein expression in the quadriceps muscle of mdx:utro mice. In these tissues, we have found ectopic expression of the cardiac isoform of the SERCA2a calcium pump predominantly expressed in the heart. Additionally, we have found expression of slow-twitch Type 1 cardiac myosin heavy chain, indicative of a fiber type switch event. We have shown that expression of both of these proteins is co-localized to specific muscle fibers. Our analyses indicate that the appearance of these proteins is directly correlated with the lowered expression of the fast-twitch calcium pump (SERCA1) and muscle fiber types (Type 2a and Type 2b).
These findings illustrate important molecular consequences of the absence of utrophin in mouse models of DMD. Identification of compensatory corrective targets may bear important therapeutic potential in muscular dystrophy and its associated cardiomyopathy.
Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart
Authors: Noack Claudia , Zafiriou Maria P , Renger Anke , Schaeffer Hans J , Bergmann Martin W , Zelaráyan Laura C 
Published: July 2021

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs.
In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation.
In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes.
CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype.
This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.
Abstract 74: Naturally Occurring Human α1a-Adrenergic Receptor Genetic Variant Induces Transformation of Cardiomyoblasts to Fibroblast-Like Phenotype
Authors: Kleine-Brueggeney Maren , Gradinaru Irina , Babaeva Ekaterina , Schwinn Debra A , Oganesian Anush 
Published: July 2021

Hypertensive heart disease is a progressive condition leading to myocardial remodeling, alterations in cardiomyocytes and fibrosis. Three α1 adrenergic receptor (AR) subtypes are expressed in human heart (α1a, α1b, α1d). Recently, we reported that human α1aAR with the naturally occurring G247R SNP (247R), originally identified in a hypertensive patient, constitutively couples to EGFR-transactivation pathway in fibroblasts and leads to hyperproliferation. We hypothesized that expression of 247R in cardiomyoblasts plays an important role in their remodeling.
We demonstrate that constitutive expression of 247R in H9C2 cardiomyoblasts changes the morphology of myoblasts to fibroblast-like cells (transformed, tr247R); higher 247R expression results in earlier onset of transformation. Expression of α1aAR-WT does not affect cell morphology. Tr247R cells exhibit 2-3 fold increased constitutive proliferation compared to control or WT cells as determined by cell counts and thymidine incorporation assays; pERK is upregulated in tr247R cells as determined by Western blot analyses. Treatment with AraC, a DNA replication inhibitor, eliminates the 247R cells but not the H9C2 myoblasts. Expression of cell-specific markers is being evaluated in tr247R and control cells. RT-PCR and zymograms revealed increased MMP2 mRNA and protein levels in tr247R cells compared to control or WT cells and are comparable to MMP2 levels in fibroblasts. Increased proliferation of tr247R cells is reduced by EGFR specific inhibitor AG1478 but not general MMP inhibitor GM6001. Agonist stimulation of tr247R results in hypertrophy as determined by leucine incorporation. Interestingly, hypertrophy but not cell proliferation is inhibited by prazosin, an α1aAR inverse agonist, suggesting that increased proliferation and hypertrophy observed in transformed cells are two independent pathways triggered by 247R.
Our data demonstrate that expression of α1aAR genetic variant triggers alterations in cardiomyoblasts via distinct signaling pathways leading to hyperproliferation, transformation of the cells to fibroblast-like phenotype and hypertrophy. Consequently, these changes may lead to fibrosis in human heart, extracellular matrix remodeling and heart failure.
Abstract 001: The Investigation of Peptide Amphiphile Micelles for Detection of Vulnerable Atherosclerotic Plaques
Authors: Chung Eun Ji , Drews Laurie , Tirrell Matthew 
Published: July 2021

Atherosclerosis is an inflammatory disease that is complicated by progressively unstable plaques which are characterized by specific molecular events. While current diagnostic imaging technology aim to determine the stability of plaques by assessing their physical attributes, molecular imaging tools aim to assess the true nature of the plaque through targeting of molecular markers. Furthermore, these existing technologies can hardly meet the sensitivity and accuracy standards that are required for any clinical application for preventive measures.
In order to preventatively intervene during the early pathology of plaque vulnerability, we are engineering monocyte-targeting, fluorescently-labeled peptide micelles (MFPMs) through the incorporation of the chemokine receptor CCR2-binding motif (residues 13-35) of the monocyte chemoattractant protein-1 (MCP-1). Since monocytes are found concentrated in plaques prone to rupture, and our molecular imaging tool binds specifically to monocytes, this nanoparticle system may provide a noninvasive, detection system for plaques of high risk.
The MCP-1 peptide or Cy-7 was conjugated to DSPE-PEG-2000 and mixed to form self-assembled, fluorescently-labeled, monocyte-targeting peptide amphiphiles. The critical micelle concentration of MFPMs was determined to be approximately 1 μM with an average diameter of approximately 15 nm, confirmed via dynamic light scattering and transmission electron microscopy. Chemotaxis assays confirmed the chemoattractive capabilities of MFPMs using murine monocytes at 4 hours in vitro, and MFPMs were confirmed to be biocompatible using murine aortic endothelial cells and smooth muscle cells after 24 hours. Peptide amphiphile micelles can be synthesized with multiple functionality (i.e. targeting element, imaging dye, etc.) and our investigations will lay the ground work for peptide amphiphile micelle-mediated drug delivery at the site of vulnerable plaques.
Abstract 137: VCAM1 and SIRPA Cell Surface Markers of Human Cardiomyocytes
Authors: Skelton Rhys J , Arasaratnam Deevina , Finnin Ben W , Haynes John M , White Anthony , Ardehali Reza , Elefanty Andrew G , Stanley Edouard G , Elliott David A 
Published: July 2021

Heart disease is the leading cause of mortality and morbidity in the Western world. The human heart has a limited regenerative capacity and there is an unmet demand for improved therapies for myocardial disease. Human stem cell derived cardiomyocytes have potential to facilitate the development of both cell based and pharmaceutical treatments. To date many studies have focused on the efficient generation of cardiomyocytes from pluripotent hESCs and iPSCs. Efficient in vitro differentiation of human cardiomyocytes is becoming a standard technique and hESC derived cardiomyocytes are beginning to be used in cardiotoxicity screens. In order to capitalise on these advances in cardiomyocyte generation and ensure the transfer of this technology into biopharmaceutical and clinical settings, enriched and well defined cell populations are required. Therefore, cell surface markers that allow the facile purification of cardiac cell lineages are needed.
This work investigates the potential of two novel cell surface markers, SIRPA and VCAM1, in isolating enriched populations of committed human cardiomyocytes. The NKX2-5GFP/w reporter line was used to characterise the temporal expression, gene expression profiles and functional properties of SIRPA and VCAM1 cell populations that are found during cardiac differentiation. We demonstrate that fully functional cardiomyocytes arise from an NKX2-5+SIRPA+ intermediate which up regulates VCAM1 concomitantly with commitment to the myogenic lineages of the heart (ie. smooth and cardiac muscle). Under the culture conditions used in this study, the NKX2-5+SIRPA+ population had a higher single cell clonogenic potential and displayed the capacity to give rise to both smooth muscle and cardiac clones. Furthermore, when cultured as re-aggregates the majority of NKX2-5+SIRPA+ cells progressed to a NKX2-5+SIRPA+VCAM1+ phenotype thus demonstrating lineage relationship of these two cell populations.
The identification of these cell surface markers provide a means for further study of linage specification during in vitro human cardiogenesis and will facilitate the development of technologies to produce well-characterised, purified human cardiomyocytes.
Abstract 174: The Origin Of Cardiac Fibroblasts During Normal Development And After Injury
Authors: Ranjbarvaziri Sara , Ali Shah R , Talkhabi Mahmood , Subat Ali , Zhao Peng , Volz Katharina S , Red-Horse Kristy , Ardehali Reza 
Published: July 2021

Fibroblasts are the most abundant cells in the heart. They play a critical role in maintaining normal myocardial function as well as mediating fibrosis that occurs upon pathological injury. However, the source of cardiac fibroblasts during development and after the injury in the adult heart remains poorly characterized. The lack of cardiac fibroblast-specific markers has limited our ability to isolate or track them during normal development and in response to cardiac injury. Here, we define cardiac fibroblasts as mesenchymal cells with a surface-marker profile of CD45-/CD31-/Ter119-/CD11b-/Thy1.1+ /Thy1.2+. We used lineage tracing, transplantation studies, and parabiosis surgeries to identify the cellular origins of cardiac fibroblasts and their contribution to fibrosis during pathological injury induced by pressure overload. We show that fibrosis is associated with proliferation of fibroblasts within 7 days after pressure overload injury with no evidence of bone marrow or hematopoietic stem cells contributing to the cardiac fibroblast population. We then used the transgenic mouse models Tie2cre;mTmG, Tbx18cre;mTmG, and Pax3cre;mTmG to lineage trace the origin of fibroblasts from the epicardium, endothelial and the neural crest, respectively. By using fluorescent activated cell sorting (FACS) and immunostaining, we demonstrate that the majority of cardiac fibroblasts are derived from the epicardium, a minority arises from endothelial cells, and a small fraction from the neural crest. Although we observed an increase in the total number of fibroblasts after pressure overload injury, this response was not restricted to any single developmental subset.
In summary, our findings show that the majority of the cardiac fibroblasts, during normal development as well as in response to pathological injury, are derived from the epicardium via epithelial-to-mesenchymal transformation. Endothelial to mesenchymal transition and neural crest sources also contribute to fibroblast generation and the progression of pressure overload-induced cardiac fibrosis.
Abstract 233: Isoproterenol-induced Cardiac Hypertrophy And Failure In Mice: Gene Network Modeling
Authors: Rau Christoph D , Wang Jessica , Ren Shuxun , Wang Zhihua , Ruan Hongmei , Wang Yibin , Lusis Aldons J 
Published: July 2021

Heart failure is highly heterogeneous and as a result, relatively few insights into the pathways and drivers of heart failure have been identified using system-wide methods such as genome-wide association studies (GWAS). We have developed a resource, the Hybrid Mouse Diversity Panel (HMDP) for high resolution GWAS and systems genetics in mice. Eight week old female mice from 93 unique inbred strains of the HMDP were given 20 μg/g/day of isoproterenol through an abdominally implanted Alzet micropump. Three weeks post-implantation, all mice were sacrificed, along with age-matched controls. The mice exhibited widely varying degrees of hypertrophy and heart functioning. A portion of the left ventricle was processed and arrayed on an Illumina Mouse Ref 8.0 platform.
We used Maximal Information Component Analysis, a novel method of network construction which allows for non-linear relationships between genes as well as non-binary partitioning of genes into sub-networks to subdivide the expression data into a series of modules. In order to identify modules which may contribute to Isoproterenol-induced hypertrophy and failure, we examined the correlation of each module to clinically relevant cardiac traits traits such as organ weights and echocardiographic parameters.
We identified several modules with strong correlations to multiple heart failure-related clinical traits, including one module of 41 genes which contained several genes of interest, including Lgals3, a diagnostic marker for heart failure. Utilizing eQTL hotspot analysis, we have identified a locus which is involved in the regulation of this module. A gene within this locus, Magi2, regulates the turnover of the β-adrenergic receptor and represents a likely candidate for the response to isoproterenol.
Abstract 234: Defining Domains In The Transcription Factor Grainyhead-like 3 Required For Protective Functions In The Endothelium
Authors: Farrokh Sabrina , Ale-Agha Niloofar , Haendeler Judith , Altschmied Joachim 
Published: July 2021

Important aspects during aging of human endothelial cells (EC) are an increased apoptosis level and a reduced migratory capacity. In a screening for anti-apoptotic genes we have identified the transcription factor Grainyhead-like 3 (GRHL3) and demonstrated its anti-apoptotic and pro-migratory effects after overexpression and knockdown in EC. To define the domains in GRHL3 responsible for these - potentially also extranuclear - functions we have cloned large scale deletion mutants and mutants with deletions of putative nuclear localization signals (NLS) and analyzed them for their intracellular localization and functional properties.
Immunostaining of transfected EC were used to examine the subcellular distribution. Two mutants with deletions in a bioinformatically predicted bipartite NLS were localized predominantly in the cytoplasm. To corroborate these data, lysates of the cells were fractionated biochemically. Western blotting confirmed the immunostaining data, indicating that we have identified the major NLS in GRHL3.
To analyze the transcriptional properties of these mutants, we constructed a GRHL3-specific luciferase reporter containing a tandem GRHL3 binding sites in front of a minimal promoter and cotransfected it with expression vectors for the GRHL3 deletion mutants. Besides the previously described activation domain we identified another region required for transcriptional upregulation of target genes in the N-terminal half of the protein.
We will now use these mutants to further dissect the regions in GRHL3 necessary for its pro-migratory and anti-apoptotic activities in endothelial cells. This will also allow us to investigate if all of these functions are mediated solely by the activation of target genes or by other mechanisms coupled to a non-nuclear function of GRHL3.