bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 260: Cardiosphere Derived Cells Generated from the Human Atria Exhibit Superior Potency Compared to the Left Ventricle
Authors: Simpson David L , Blusztajn Agnieszka , Valle Ileana , Kreke Michelle , Marban Linda , Smith Rachel R
Published: July 2021

Currently, allogeneic cardiosphere-derived cells (CDCs) are being tested in a phase I/II clinical trial. Our manufacturing process utilizes the atria and other regions of the heart to build a master cell bank, but leaves the left ventricle (LV) intact thus eliminating a potential source to build our clinical stocks. In an effort to increase CDC yield per heart, we compared CDCs generated from the atria to the left ventricle (LV). To investigate this hypothesis we characterized CDCs by flow cytometry, growth kinetics and in vivo potency. The surface marker profiles for atrial CDCs and LV CDCs were very similar. Of the 17 markers used to characterize CDCs (c-kit, MDR-1, Sca-1, Abcg2, CD133, CD31, CD34, CD45, CD105, CD29, CD44, CD73, CD166, CD140b, CD90, DDR2 and α-SMA) only one demonstrated substantial differential expression in LV versus atria. PDGFR-β (CD140b) was upregulated in CDCs derived from the LV compared to the atria. Consistent with this finding, GEO2R analysis of DNA microarray data revealed increased PDGFR-β and PDGF-B expression in normal human LV tissue compared to atrial tissue. We have also observed that CDC expression of PDGFR-β negatively correlates with in vivo potency (R=0.899) suggesting that the high expression of this marker in CDCs derived from the LV may limit its regenerative performance. Indeed when calculated growth rates were compared, tissue samples from the atria yielded 4 to 6-fold more CDCs compared to the LV (atria=26±10 versus LV=5±5 Million CDCs/g/day). Also, CDCs derived from the atria and LV led to differential improvements in ejection fraction (EF) over three weeks. CDCs from the atria significantly outperformed the control group (atrial CDCs=Δ+12.6±10.8%, control=Δ-15.3±13.6%), while CDCs from the LV showed minimal treatment effects and failed to meet our minimal potency requirement (LV CDCs=Δ+0.0±6.2%). In conclusion, LV CDCs display limited potential for clinical use. This observation provides a unique opportunity to explore the mechanisms that govern functional potency and assist in understanding the basic processes involved in CDC mediated repair.
Abstract 270: Role of the Mitochondrial Iron in Cardiomyoblast Survival and Maturation of Cytosolic Fe/S Proteins.
Authors: Thakur Anita , Bayeva Marina , Ardehali Hossein
Published: July 2021

ATP-binding cassette (ABC)-B8 is an ABC half transporter that resides in the inner mitochondrial membrane. We previously showed that ABCB8 has a role in mitochondrial iron export, and that a reduction in ABCB8 both in vitro and in vivo results in mitochondrial iron accumulation, increased reactive oxygen species (ROS) and cell death, and decreased activity of cytosolic Fe/S proteins. However, it is not known whether the cytotoxic effects of ABCB8 knockdown are due to mitochondrial iron accumulation or decreased cytosolic Fe/S protein or other processes. Furthermore, the link between mitochondrial iron and the activity of cytosolic Fe/S proteins is uncharacterized. Here, we studied whether a reduction in mitochondrial iron can reverse the effects of ABCB8 knockdown on cell survival, ROS production and the activity of Fe/S proteins. We altered the mitochondrial iron using various iron chelators and by decreasing the levels of mitochondrial iron importer, mitoferrin-2 (MFRN2). The increase in mitochondrial iron and ROS levels associated with ABCB8 knockdown was significantly reversed by iron chelators and with MFRN-2 knockdown. Furthermore, cell death was also reversed with iron chelators and MFRN-2 knockdown, suggesting that the cytotoxic effects of ABCB8 knockdown is due to mitochondrial iron accumulation. We then studied the effects of iron chelators on cytosolic Fe/S proteins. Iron chelators reversed the defect in cytosolic Fe/S proteins that is associated with ABCB8 knockdown in cell culture or knockout in mice. However, antioxidants and knockdown of MFRN-2 failed to have similar effects. These results indicated that the defect in the maturation of cytosolic Fe/S proteins due to mitochondrial iron accumulation can be reversed by chelating mitochondrial iron, and is independent of the associated oxidative stress. Thus, mitochondrial iron levels are likely sensed in the cytoplasm and determine the maturation of cytosolic Fe/S proteins. Altogether, these studies provide insights into the role of mitochondrial iron in the cytotoxic effects of ABCB8 knockdown, and suggest that mitochondrial iron is sensed in the cytoplasm and influences the maturation of cytosolic Fe/S proteins.
Abstract 291: Fibulin-1 is Required to Prevent Hypoplastic Semilunar Valve Formation
Authors: Harikrishnan Keerthi , Cooley Marion A , Twal Waleed O , Fresco Victor M , Kern Christine B , Argraves W. Scott
Published: July 2021

Fibulin-1 (Fbln1), an extracellular matrix protein, is prominently expressed during outflow tract (OFT) morphogenesis as a component of both the endocardial cushions and OFT valves. Here we sought to investigate the role of Fbln1 in the process of OFT valvulogenesis. Since Fbln1 null mice die perinatally, we first evaluated the effects of Fbln1 deficiency on OFT valve morphology in hearts of embryos at E17.5. Morphometric analysis of 3D reconstructions revealed that in Fbln1 nulls the volume of the anterior leaflet of the pulmonary valve, and the left and non-coronary leaflets of the aortic valve were significantly reduced as compared to wild type (WT) (30-40% reduction; WT n=6, null n=8; p<0.05). The reduced leaflet size correlated with decreased leaflet cell numbers (45%, 30% and 30% respectively; p<0.05). TUNEL and BrdU analysis showed no difference between E17.5 WT and Fbln1 null OFT valves. To determine when abnormalities of OFT valve leaflet cell numbers first occurs, OFT endocardial cushions and prevalvular mesenchyme from E10.5-14.5 null and WT embryos were analyzed. At E10.5, the proximal OFT cushions of Fbln1 nulls were hypercellular (34% increase; p<0.05; n=5 for each genotype) whereas cellularity of distal cushions was not different from WT (p= 0.49). The fact that the ratio of proximal OFT cushion mesenchymal cells to endothelial cells was increased in E10.5 Fbln1 null cushions suggests that alteration in EMT might contribute to the hypercellularity. At E12.5, cellularity of the proximal OFT cushions was observed to decrease by 35% (p<0.05; WT n=4; null n=4). This was accompanied by a 172% increase in apoptosis, with no alteration in the level of proliferation (p<0.05; WT n=3; null n=4). Decreased cellularity and increased apoptosis persisted in the OFT prevalvular mesenchyme of Fbln1 nulls at E14.5 (p<0.05; WT n=2; null n=2). Taken together, Fbln1 specifically regulates cellularization of the proximal endocardial cushion and prevalvular mesenchyme. Mechanistically, Fbln1 may act as a suppressor of cushion EMT and indirectly or directly as an inhibitor of apoptosis.
Abstract 293: Tcf7l2 Expression And Isoform Switching In Mouse Hearts
Authors: XIAO LU , Bai Haiqing , Boyer James , Ye Bo , Hou Ning , Xu Haodong , Li Faqian
Published: July 2021

Lu Xiao, Haiqing Bai, James Boyer, Bo Ye, Ning Hou, Haodong Xu, and Faqian Li Department of Pathology and Laboratory Medicine and Cardiovascular Research Institute, University of Rochester Medical Center, Rochester, NY, USA

Backgrounds:
Canonical Wnt signaling appears to have multiphasic and often antagonistic roles in cardiac development. The molecular mechanism for these opposing actions is not clear. We hypothesized that alternative splicing of TCF7L2, a nuclear interaction partner of beta-catenin is involved in the specificity of canonical Wnt signaling.

Methods:
RT-PCR were performed on embryonic (E16.5) and neonatal (day 8) hearts with primers spanning the end of first exon and the beginning of last exon and the products were cloned and sequenced.

Result:
There are totally 18 exons identified so far in TCF7L2. We sequenced 56 clones and 53 clones (29 from day 8) and (24 from E16.5) contained TCF7L2 sequences. No exon 6 or exon 17 was found in TCF7L2 transcripts of mouse hearts. Most clones (more than 80%) from E16.5 and day 8 hearts excluded exon 4. Both E16.5 and day 8 hearts had one clone with exon 9 deletion which does not change reading frame and another with alterations in exon 3 that lead to reading frame shift and premature stop codon. As reported in other organs, there were extensive alternative splicing in the C-terminal exons 14, 15 and 16. The inclusion of exon 14 was more frequently in day 8 (18 of 29, 62%) than in E16.5 (8 of 24, 33%) hearts. The peptide encoded by exon 14 has conserved functional motif. Additionally, this alternative exon usage can change the C-terminus of TCF7L2 to include or exclude the so-called E tail with two binding motifs for C-terminal binding protein.

Conclusion:
The isoform switch of TCF7L2 occurs in neonatal mouse hearts and may have a role in the terminal differentiation of cardiac myocytes during this period.
Abstract 295: Enhanced Cardiac Function And Lower Circulating Aldosterone Levels In βArrestin1 Knockout Mice During Post-myocardial Infarction Heart Failure Progression
Authors: Lymperopoulos Anastasios , Rengo Giuseppe , Gao Erhe , Siryk Ashley , Dabul Samalia , Koch Walter J
Published: July 2021

Introduction:
Chronic heart failure (HF) is characterized by enhanced circulating cardiotoxic hormones, among the most prominent of which is aldosterone, which contributes to the increased morbidity and mortality of the disease by promoting cardiac adverse remodeling post-myocardial infarction (MI). Cardiac β-adrenergic receptor (ΑR) desensitization and downregulation are a hallmark abnormality in HF at the molecular level and are due to the concerted action of cardiac G protein-coupled receptor kinase-2 (GRK2), together with its co-factors in receptor desensitization, the βarrestins (βarrs). We have also recently established that βarr1 promotes angiotensin II-dependent aldosterone production in the adrenal cortex, and this leads to elevated circulating aldosterone levels in vivo, both under normal conditions and during post-MI HF progression.

Hypothesis:
Herein, we sought to investigate the effects of genetically deleting βarr1 on post-MI cardiac function and hyperaldosteronic status in mice progressing to HF.

Methods:
We uitilized the βarr1KO mouse model and studied these mice at 4 weeks after surgically induced MI, in parallel with C57/B6 wild type (WT) controls. Cardiac function was assessed by echocardiography and in vivo catheterization. Plasma aldosterone was measured by ELISA.

Results:
Cardiac function is markedly improved in βarr1KO`s at 4 weeks post-MI, as evidenced by increased ejection fraction compared to WT mice (41.5 + 2.8 % vs. 21.8 + 2.4 %, respectively, n=9, p<0.0001) and increased isoproterenol-induced contractility. Additionally, cardiac dimensions are significantly reduced compared to WT`s, indicating attenuation of adverse cardiac remodeling. Importantly, plasma circulating aldosterone levels are significantly lowered and cardiac βAR signaling and function appear elevated in post-MI βarr1KO`s compared to control WT`s.

Conclusions:
Genetic deletion of βarr1 substantially improves cardiac function, adverse remodeling, hyperaldosteronism, and cardiac βAR function during post-MI HF progression. The underlying mechanism is attenuation of both cardiac βAR desensitization/downregulation and adrenal aldosterone production, which is βarr1-dependent.
Abstract 297: Cardiac Transforming-growth-factor β-stimulated Clone 22 Gene Expression By Hypertrophic Stimuli
Authors: Kelloniemi Annina , Aro Jani , Koivisto Elina , Ruskoaho Heikki , Rysä Jaana
Published: July 2021

Objectives:
Transforming-growth-factor β-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains largely unknown. The aim of the present study was to characterize the cardiac TSC-22 expression.

Methods:
Acute pressure overload was accomplished in conscious Sprague-Dawley (SD) rats by intravenous infusion of arginine
8
-vasopressin (AVP, 0.05 μg/kg/min) for 4 hours and subcutaneous infusion of angiotensin II (Ang II, 33 μg/kg/h) with and without Ang II receptor type 1 blocker losartan (400 μg/kg/h) by using osmotic minipumps for 2 weeks. Adenovirus-mediated intramyocardial gene transfer of TSC-22 was performed into left ventricle (LV) of SD rats. Experimental myocardial infarction (MI) was produced by ligation of the left anterior descending coronary artery. Cultured neonatal rat ventricular myocytes (NRVM) were treated with endothelin-1 (ET-1, 100 nM).

Results:
A significant 1.6-fold increase (
P
<0.05) in LV TSC-22 mRNA levels was noted already after 1 hour AVP infusion. Moreover, Ang II infusion markedly upregulated TSC-22 expression, LV mRNA levels being highest at 6 hours (11-fold,
P
<0.001). Simultaneous infusion of losartan completely abolished Ang II-induced increase in TSC-22 mRNA levels. Adenovirus-mediated gene transfer of TSC-22 into LV resulted a 1.9-fold (
P
<0.001) increase in TSC-22 mRNA levels, accompanied by upregulated BNP mRNA levels (1.4-fold,
P
<0.01). In response to experimental MI, TSC-22 mRNA levels were elevated 4.1-fold (
P
<0.001) at 1 day and 1.9-fold (
P
<0.05) at 4 weeks. In cultured NRVM, ET-1 treatment increased TSC-22 mRNA levels from 1 h to 24 h, the greatest increase being observed at 12 h (2.7-fold,
P
<0.001). TSC-22 protein levels were upregulated from 4 h to 24 h with the highest increase at 24 h (4.7-fold,
P
<0.01).

Conclusion:
These results indicate that TSC-22 expression is rapidly activated in response to pressure overload, MI and in ET-1 treated cultured NRVM. Moreover, adenovirus-mediated overexpression of TSC-22 mRNA was associated with elevated left ventricular BNP mRNA levels.
Abstract 307: Fetal Deiodinase Increases in a Rodent Model of Myocardial Infarction
Authors: Henderson Kyle K , Vuppuluri Rohit , Murphy Robert
Published: July 2021

Rationale:
Thyroid hormone (T
3
) has been shown to play a critical role in myocardial vascular homeostasis. Importantly, patients with congestive heart failure frequently have reduced serum T
3
concentrations which may induce vascular rarefaction and/or limit angiogenesis during left ventricular remodeling. One possible mechanism for reducing T
3
concentration is the reactivation of Type III deiodinase. Type III deiodinase (D3) is a fetal enzyme that inactivates thyroxine as well as T
3
.

Objective:
Determine whether enzymes regulating myocardial thyroid hormone concentration are altered after a myocardial infarction.

Hypothesis:
Myocardial D3 protein levels will increase in a rodent model of myocardial infarction, and immediate T
3
supplementation will augment angiogenesis and adaptive left ventricular remodeling.

Methods and Results:
The left anterior descending coronary artery was ligated in adult male Sprague Dawley rats with controls undergoing identical procedures without ligation. Osmotic mini-pumps were immediately implanted subcutaneously to deliver saline or T
3
(10ug/kg/day). One week post-surgery, hearts were removed and subdivided into ischemic, border-zone, and non-infarcted/posterior wall. Results show that Type II deiodinase (D2) protein levels were increased in the ischemic zone of infarcted hearts (D2 is normally expressed in the heart and converts thyroxine into T
3
). D3 protein levels were also increased in the ischemic zone of MI-hearts. Importantly, the D2 to D3 ratio was reduced by ~60% suggesting T
3
concentrations could be reduced in the ischemic regions of the heart. Additionally, D3 dimers were only present in the ischemic region of the MI heart. Because dimerization is thought to be required for enzymatic activity, ischemic areas of the heart may have regional hypothyroidism. Overall, these findings support the hypothesis that myocardial D3 protein levels are increased 7-days after a myocardial infarction.

Conclusion:
The increase in dimerized D3 protein levels in the ischemic region of the heart may reduce local T
3
concentrations and contribute to mal-adaptive left ventricular remodeling.
Abstract 328: Med13 regulates cardiac metabolism
Authors: Baskin Kedryn K , Grueter Chad E , Kusminski Christine M , Scherer Philipp E , Bassel-Duby Rhonda , Olson Eric N
Published: July 2021

Background:
The heart is a metabolic organ that primarily utilizes fatty acids as energy substrate. While it is well established that the heart is metabolically flexible, the transcriptional network regulating cardiac metabolism is only partially understood. We have previously demonstrated that cardiac overexpression of Med13, a component of the Mediator Complex that regulates transcription, results in a lean phenotype with enhanced basal metabolic rates. We now investigate the mechanisms contributing to metabolic changes in mice with cardiac over-expression of Med13(Med13cTg).

Methods and Results:
Cardiac fludeoxyglucose (18F-FDG)-PET imaging analysis revealed that Med13cTg hearts take up more glucose than wild type littermates. To determine pathways responsible for enhanced glucose uptake, ventricles from Med13cTg mice were subjected to RNA-seq and metabolomic analysis. The expression of fatty acid oxidation genes was decreased in Med13cTg hearts, accompanied by an increase in acyl CoA and a decrease in acetyl CoA. These data suggest that beta oxidation is decreased in Med13cTg hearts. Mitochondria function was therefore determined in Med13cTg hearts by performing electron-flow analyses and assessing oxygen consumption rates. Indeed, oxygen consumption rates were decreased in mitochondria isolated from Med13cTg hearts. Expression of Krebs Cycle genes and corresponding intermediary metabolites were also decreased in Med13cTg hearts, suggesting decreased flux through this pathway as well.

Conclusions:
Overexpression of Med13 in the heart increases glucose uptake and decreases fatty acid oxidation in the heart. We speculate that Med13 transcriptionally regulates key mediators of cardiac metabolism. The mechanisms by which this occurs are currently under investigation.
Abstract 331: Cardiac Stem Cell Therapy in Conjugation with Anti-inflammatory Small molecule enhances function of infarcted rat hearts.
Authors: Sharma Sudhish , Simpson David , Mishra Rachana , Deshmukh savitha , Kaushal Sunjay
Published: July 2021

The transplantation of human cardiospheres derived cells (hCDCs) in clinical Phase I trials involving adult ischemic patients, results in reduction in scar formation. But this therapy faces three major issues: low survival rate (1-5%) of the transplanted cells and limited ventricular ejection fraction improvement. One of the major causes of decreased hCDCs engraftment is the prolonged and sustained pro-inflammatory response after acute myocardial infarct (MI). Pro-survival cocktail has been used to overcome this problem but it has major disadvantages for human use as a multi-component mixture, it may change the function of the stem cells and this method depends on anti-apoptotic molecules which may have side effects on the overall cellular turnover. A drug screen in our laboratory has discovered potent anti-inflammatory small molecules, Celastrol (CEL), a quinone triterpene. Oral administration of CEL significantly reduced the MI induced inflammatory cytokines release IL-1β by 2.6±0.4 fold (p<0.05, n=5), IL-6 by 2.1±0.2 fold, (p<0.01, n=5), TNF-α by 3.2±1.1 fold, (p<0.001, n=5) and MMP2 1.5±0.2, (p<0.05, n=5:) as analyzed in rat tissue by RT-PCR. This correlates with improved ventricular ejection fraction at day 7 and day 28 post MI (CEL 45.8 ± 2.4% vs. CNT, 34.8±3.3%, p=0.005 (7d), and 44.3 ± 2.8% vs 34.4 ± 4.1%, p=0.01 (28d). After 28 days post MI, MI+CEL group had a higher percentage of viable myocardial tissue as compared to MI group (MI+CEL, n=4: 70.2 ± 0.5%, vs MI, n=5: 56.38 ± 3.8%, n=4 p<0.05) as analyzed Masson’s Trichrome staining. We next combined Celastrol therapy with cardiac stem cell therapy (pretreatment of CDCs for 24 hours with Celastrol prior to their injection in infarcted myocardium) and evaluate the potential of CEL treated hCDCs to repair/preserve infarcted myocardium by ventricular ejection fraction. Echos performed after LAD ligation revealed a preserved ventricular ejection fraction in rats treated with CEL+hCDCs compared to controls (IMDM media) or CDCs by itself. (hCDCs+CEL, n=4: 62.9±1.0% vs IMDM, n=4: 32.95±2.7%, P<0.05; hCDCs, n=4: 53.28±4.0%, P<0.05 at 28 days). These initial studies suggests the effectiveness of combinatorial therapy of CEL and hCDCs. Oral delivery makes CEL a potentially clinically useful adjunct after MI.
Abstract 337: cAbl Tyrosine Kinase Increases Catalase Activity in the Catalase-Dysregulated Newborn Rabbit Heart
Authors: Cabigas E Bernadette , Liu Jie , Boopathy Archana , Che Pao Lin , Crawford Brian , Baroi Gitanjali , Wagner Mary B , Davis Michael E
Published: July 2021

Introduction:
Pediatric patients undergoing corrective cardiothoracic surgery numbered in the tens of thousands annually and therefore it is necessary that pre- and post-operative strategies be employed to limit ischemia-reperfusion (IR) injury. We have found differences in the way the newborn (versus the adult) heart responds to IR injury. In the adult, catalase activity increases appropriately with increasing levels of hydrogen peroxide (H2O2), conferring cardioprotection. This effect however does not occur in the newborn.

Materials and Methods:
Hypoxia studies were carried out using primary cardiomyocytes from adult (>8 weeks) and newborn rats. Ischemia-reperfusion (IR) studies were performed using newborn (4-5 days) rabbits. Isolated rat cardiomyocytes were exposed to 1 hour of hypoxia (95%N2,5%CO2). Immunoprecipitation and western blot analysis were done for phosphorylated and total catalase and c-Abl. In addition, catalase activity was determined spectrophotometrically by measuring the decomposition of H2O2. Newborn rabbit hearts were perfused using Langendorff method with ischemia and reperfusion periods lasting 30 and 60 minutes, respectively. DPH (200µM), a cAbl activator was injected directly into the newborn right ventricle (RV) two minutes before the reperfusion period.

Results:
Following hypoxia, the ratio of phosphorylated to total catalase and c-Abl in isolated newborn rat myocytes did not increase and were significantly lower (1.92- and 6.21-fold, respectively; p<0.05) than their adult counterparts. Interestingly, when DPH was administered one hour prior to hypoxia, not only was catalase activity significantly increased 6.28-fold (p<0.01) but phosphorylation levels were also altered. Finally, direct injection of DPH into the RV of Langendorff-perfused newborn rabbit hearts caused a significant improvement in percent recovery of developed pressure (from 49.53±5.42% to 125.5±15.5%) after IR, a key indicator of cardiac function. Catalase activity in these same hearts increased 2.92-fold.

Conclusion:
In the newborn, decreased phosphorylation of catalase by c-Abl potentially mediates IR-induced dysfunction. Activation of c-Abl may be a strategy to prevent ischemic injury associated with surgical procedures.
Abstract 341: Nucleotide Triphosphate Diphosphohydrolase-1 (NTPDase1) Overexpression Improves Post-Myocardial Infarction Recovery
Authors: Gao Erhe , He Heng , Zhang Bo , Wheeler Debra G , Mahamud Shouvik D , Dwyer Karen M , Robson Simon C , Cowan Peter J , Koch Walter J , Gumina Richard J
Published: July 2021

Background:
With myocardial injury extracellular levels of adenosine triphosphate (ATP) and adenosine diphosphate (ADP) increase. These extracellular ATP and ADP levels are modulated via hydrolysis by triphosphate diphosphohydrolase-1 (NTDP-1/CD39) to adenosine monophosphate (AMP) subsequently converted by ecto-5’-nucleotidase (CD73) to adenosine. Previous data demonstrated significantly smaller infarcts in mice globally overexpressing NTPDase1. The current objective was to determine whether global overexpression of NTPDase1 would beneficially or detrimentally affect post-infarction myocardial recovery.

Methods:
Myocardial infarction was induced by permanent coronary artery ligation in transgenic mice overexpressing human NTPDase1 driven by the murine H-2Kb (MHC class I) promoter in mice. Survival, myocardial function and BNP were measured to assess the effect on post-infarction myocardial recovery.

Results:
Following permanent coronary artery ligation there was decreased mortality in NTPDase1-Tg animals compared to WT animals (Fig A). Echocardiographic assessment of ejection fraction revealed that compared to baseline the post-MI EF of NTPDase1-Tg mice was not significantly diminished (Fig. B). Consistent with preserved cardiac function, plasma levels of brain natriuretic peptide (BNP) were significantly reduced in NTPDase1-Tg mice versus WT littermate control mice at post-MI day 28 (Fig. C).

Conclusions:
Overexpression of NTPDase1 improves post-infarction myocardial recovery.
Abstract 353: Hdac Class I Inhibition Diminished Cd90+ Cardiac Fibroblast Activation Via Gsk3b/β-catenin Pathway.
Authors: Nural-Guvener Hikmet , Zakharova Luidmila , Mastroeni Diego , Gaballa Mohamed
Published: July 2021

Myofibroblast activation is one of the major mechanisms of scar formation and interstitial myocardial fibrosis after myocardial infarction (MI) and subsequent heart failure. Elucidating the mechanisms of myofibroblast activation will provide a strategy to treat heart failure. Histone Deacetylases (HDAC) were reported to regulate myofibroblast differentiation and extracellular matrix deposition in lung, kidney and liver fibrosis. Furthermore, HDAC inhibitors were shown to retard myocardial remodeling, interstitial fibrosis and improve cardiac function after MI. However, the mechanisms are unclear. Here we examined whether HDAC1/2 are up-regulated after MI, identify which myocardial cell type is predominately expressing HDAC, and examine the effects of HDAC inhibition on myocardial fibrosis. MI was created in rats by ligation of the left coronary artery. We found that, in sham rats, HDACs were uniformly expressed in both right and left ventriculars. On the other hand, we observed up-regulation of both HDAC1 and 2 in the infarcted LV following MI. In addition, HDAC1 and 2 were co-localized with cardiac fibroblast markers CD90, Vimentin, and αSMA in the infarcted area. Treatment of CD90+ cells isolated from both CHF and sham atrial culture with pan HDAC inhibitor (100 nM, or 300 nM) TSA diminished cardiac fibroblasts activation as indicated by the decrease in αSMA and the increase in E-cadherin gene expressions. Similarly, treatment of CD90 cells with a specific HDAC class I inhibitor (2 μM, Mocetinostat) upregulated E-cadherin and diminished the markers of fibrosis (αSMA, MMP-2, and Collagen III).The decrease in fibroblast activation by HDAC inhibition was associated with increase in p-Gsk3β/Gsk3β ratio and β-catenin protein content. In addition, Mocetinostat treatment of CD90+ cells up-regulated cleaved-Caspase3, an indicator of apoptosis. Taken together, diminishing cardiac fibroblast activation using HDAC class I inhibitor appears to be dependent on Gsk3β/β-catenin pathway.
Abstract 357: Branched-Chain Amino Acid Metabolic Reprogramming in Heart Failure
Authors: Sun Haipeng , Olson Kristine , Zhou Meiyi , Prosdocimo Domenick , Gao Chen , Wang Zhihua , Jeyaraj Darwin , Youn Ji-Youn , Ren Shuxun , Chang Ching-Pin , Cai Hua , Ping Peipei , Schulze Christian , Lynch Christopher , Jain Mukesh , Wang Yibin
Published: July 2021

Metabolic remodeling is an integral part of heart failure. Current studies are largely focusing on glucose and fatty acid metabolism, while little is known about the changes in amino acid homeostasis during heart failing process. Branched chain amino acids (BCAAs), including leucine, isoleucine, and valine, serve as not only essential building blocks for protein synthesis, but also important energy source and signaling molecules that have significant effects on cell growth and function. In this study, we demonstrated that the BCAA catabolic intermediate branched-chain keto acid (BCKA) accumulated in both mouse and human failing heart. BCAA catabolic genes were selectively and significantly down-regulated at both mRNA and protein levels in failing heart in mice, mimicking a similar expression pattern observed in neonatal heart. Using both
in vitro
and
in vivo
models, we established that BCAA catabolic genes were regulated by Krüppel-like factor 15 (KLF15), a key transcriptional regulator for glucose, fat, and amino acid nutrient homeostasis, suggesting that the KLF15-mediated BCAA catabolic regulation is part of the metabolic remodeling during heart failure. Genetic ablation of PP2Cm, a key regulator of BCAA catabolism, led to a significant impairment of BCAA catabolic activities and accumulation of BCKA in cardiac tissue. Importantly, PP2Cm deficiency accelerated heart failure under pressure overload. PP2Cm deficiency or elevated BCKA induced oxidative stress in cardiomyocytes and impairment of oxygen consumption and ATP production of mitochondria. Antioxidant treatment ameliorated the heart failure progression in PP2Cm deficient animals. Taken together, our data established for the first time that BCAA catabolic reprogramming is an integral component of metabolic remodeling during heart failure, and this remodeling can significantly contribute to heart failure progression.
Abstract 100: G-Protein Coupled Estrogen Receptor1 Prevents Mitochondria Inner Membrane Protein (Mitofilin) Degradation after Ischemia/Reperfusion
Authors: Harris Tanoya L , Olde Bjorn , Leeb-Lundberg Fredrik L , Bopassa Jean C.
Published: July 2021

We recently found that estrogen treatment delays the mitochondrial permeability transition pore opening and reduces ROS production after ischemia/reperfusion, suggesting that estrogen promotes mitochondrial integrity. As mitochondrial inner membrane protein (mitofilin) has been found to control mitochondrial cristae morphology and function, we investigated whether estrogen effect on mitochondrial integrity after ischemia/reperfusion involved regulation of mitofilin via activation of G-Protein Coupled Estrogen Receptor1 (GPER1).

Isolated hearts from male WT (C57BL/6NCrL), and GPER1-/- mice were perfused using Langendorff technique, with and without estrogen (40 nM). Hearts were subjected to 20 min global ischemia followed by 10 min reperfusion. Mitochondria were isolated, and 2D-DIGE followed by mass spectrometry was performed. Mitofilin expression level was confirmed using Western blot analysis in mitochondrial fractions. Mitofilin distribution was visualized using confocal microscopy in isolated cardiomyocytes, and its spatial organization in mitochondria was imaged using high resolution fluorescence microscopy. Electron microscopy was used to observe mitochondrial cristae morphology.

We obtained 52 unique proteins of interest, in which mitofilin was identified. Immunoblot analysis confirmed the increased in mitofilin level with estrogen treatment as compared to control in WT but not in GPER1 KO. Cardiomyocyte images revealed as observed in non-ischemic myocytes that estrogen treatment conserved mitofilin distribution in the peri-membrane and T-tubules, while only peri-membrane mitofilin was more visible in control group. High resolution microscopy showed a better spatial organization of mitofilin in single mitochondria with estrogen treatment compared to control, in which mitofilin was almost absent. Electron microscopy showed that mitochondrial morphology was conserved in estrogen-treated group as cristae were well organized compared to control, in which cristae were disrupted.

These data indicate that estrogen action induces regulation of mitofilin expression during ischemia/reperfusion. Estrogen effect on mitofilin may contribute to improved mitochondrial integrity and function.
Abstract 104: Sex-specific Impact Of S-nitrosoglutathione Reductase (gsnor) On Ventricular Remodeling And Function Following Myocardial Infarction In Mice.
Authors: Kulandavelu Shathiyah , Paulino Ellena , Takeuchi Lauro , Kanashiro-Takeuchi Rosemeire , Balkan Wayne , Hare Joshua M
Published: July 2021

Introduction:
Female hearts are less susceptible to myocardial injury following myocardial infarction (MI) and estrogen working through the nitric oxide (NO) pathway is thought to play a role. S-nitrosylation of cysteine thiols is a major signaling pathway through which NO exerts its action and mice with targeted deletion of S-nitrosoglutathione reductase (GSNOR), a denitrosylase that regulates S-nitrosylation, show increased levels of nitrosylated proteins. Male GSNOR
-/-
mice show a more favorable outcome after MI as compared to wild-type (WT), including reduced myocardial infarct size, improved ejection fraction and preserved left ventricular volumes. Whether female GSNOR
-/-
mice show gender-related cardiac protection following MI was not known and thus investigated.

Methods & Results:
MI was induced in male and female GSNOR
-/-
mice and their respective controls, C57Bl/6J, at 3-5 months via left anterior descending coronary artery occlusion. Serial echocardiography was performed prior to MI and after 1- and 4-weeks post-MI to assess ejection fraction (EF) and left ventricular volume both at diastole (end-diastolic volume, EDV) and systole (end-systolic volume, ESV). Compared to WT, GSNOR
-/-
males showed less dilation in both EDV (98.6 ± 9.3 mm vs. 140.6 ± 7.4 mm in WT, P<0.001) and ESV (73.1 ± 9.2 vs. 112.7 ± 7.3 mm in WT, P<0.05) at 4 weeks post-MI. Whereas, GSNOR
-/-
females showed greater dilation in both EDV (162.2 ± 13 vs. 83.8 ± 12 mm in WT, P<0.001) and ESV (141.8 ± 13 vs. 65.6 ± 12 mm in WT, P<0.001) as compared to WT females. EF decreased (P<0.001) in all groups post-MI, but at 4 weeks post-MI, it was significantly worse in GSNOR
-/-
females compared to GSNOR
-/-
males (14 ± 4% vs. 28 ± 3%, P<0.05).

Conclusion:
GSNOR
-/-
females exhibit significantly lower EF and greater dilation of left ventricular volumes both at diastole and systole following MI than any of the other groups suggesting that S-nitrosylation plays an important role in gender-related cardiac protection following myocardial injury. These findings suggest the importance of taking gender into account when exploring novel therapeutic treatments for myocardial injury.
Abstract 115: S100A6 Regulates Endothelial Cell Cycle Progression via Modulation of the STAT1 Signaling Pathway
Authors: Lerchenmüller Carolin , Heissenberg Julian , Peppel Karsten , Katus Hugo A , Busch Hauke , Börries Melanie , Most Patrick
Published: July 2021

S100A6, also known as Calcyclin, is a 10.5kDa Ca
2+
-binding protein that belongs to the S100 protein family. We have found S100A6 to be highly expressed throughout all vascular layers during the remodeling process in pig coronary arteries after stenting and rat carotid arteries post balloon dilatation injury. It was the objective of this study to decipher S100A6’s function in vascular cells.

Abundant S100A6 expression was confirmed
in vitro
in human umbilical vein endothelial cells (HUVEC) and human umbilical artery smooth muscle cells (SMC). Upon serum stimulation, a significant increase of S100A6 protein levels was observed in both cell types. S100A6 depletion due to siRNA transfection lead to a profoundly reduced proliferation rate in response to growth factors (e.g. VEGF-A), shown by stagnating cell counts and decreased EdU incorporation in HUVEC and SMC. Of note, S100A6 depletion caused a significant migration deficit of SMC. Also, reduced S100A6 levels in HUVEC lead to an increase in cellular senescence, as measured by the expression of senescence associated β-galactosidase expression. To decipher the molecular mechanisms that lead to the phenotype of S100A6 depleted vascular cells, a time-resolved gene expression analysis was carried out in HUVECs and revealed S100A6 to be in control of antiproliferative signaling pathways, while typical pro-proliferative signaling, e.g. the MAPK pathway, was not disrupted. S100A6 depletion caused increased expression and activation of the STAT1 signaling pathway. With increased STAT1 activity, interferon-inducible protein (e.g. IFITM1) expression was upregulated and lead to stabilization and increased expression of the p53 pathway, specifically p21. We propose that S100A6, found to co-localize with the VEGFR2 in a Ca
2+
-dependent manner, controls antiproliferative signaling by inhibition of the STAT1-phosphorylation by the VEGFR2 and suppression of the STAT1-p53 signaling cascade in human endothelial cells. Endothelialization is a hallmark of vascular healing after stenting and balloon dilatation, for example, while it is of utmost importance to block neointima formation. Understanding these signaling pathways is critical to better direct optimization of interventional therapies.
Abstract 116: Arterial Endothelial Cell Has Stronger Angiogenic Potential Compared To Venous Endothelial Cell Through Up-regulation Of Endogenous FGF2 And FGF5 Expression In 3D Microfluidic System
Authors: Seo Ha-Rim , Jeong Hyo Eun , Joo Hyung Joon , Choi Seung-Cheol , Kim Jong-Ho , Park Chi-Yeon , Choi Ji-Hyun , Cui Long-Hui , Chung Seok , Lim Do-Sun
Published: July 2021

Background:
Human body contains many kinds of different type of endothelial cells (EC). However, cellular difference of their angiogenic potential has been hardly understood. We compared
in vitro
angiogenic potential between arterial EC and venous EC and investigated its underlying molecular mechanisms.

Method:
Used human aortic endothelial cells (HAEC) which was indicated from arterial EC and human umbilical vein endothelial cells (HUVEC) indicated from venous EC. To explore angiogenic potential in detail, we adopted a novel 3D microfluidic angiogenesis assay system, which closely mimic in vivo angiogenesis.

Results:
In 3D microfluidic angiogenesis assay system, HAEC demonstrated stronger angiogenic potential compared to HUVEC. HAEC maintained its profound angiogenic property under different biophysical conditions. In mRNA microarray sorted on up- regulated or down-regulated genes, HAEC demonstrated significantly higher expression of gastrulation brain homeobox 2 (GBX2), fibroblast grow factor 2 (FGF2), FGF5 and collagen 8a1. Angiogenesis-related protein assay revealed that HAEC has higher secretion of endogenous FGF2 than HUVEC. HAEC has only up-regulated FGF2 and FGF5 in this part of FGF family. Furthermore, FGF5 expression under vascular endothelial growth factor-A (VEGF-A) stimulation was higher in HAEC compared to HUVEC although VEGF-A augmented FGF5 expression in both HAEC and HUVEC. Those data suggested that FGF5 expression in both HAEC and HUVEC is partially dependent to VEGF-A stimulate. HUVEC and HAEC reduced vascular density after FGF2 and FGF5 siRNA treat.

Conclusion:
HAEC has stronger angiogenic potential than HUVEC through up-regulation of endogenous FGF2 and FGF5 expression
Abstract 120: Genome-wide DNA Methylation Analysis Reveals Dynamic Changes in the Cardiac Methylome During Post-natal Heart Development
Authors: Sim Choon Boon , Ziemann Mark , Harikrishnan K.N. , Kaspi Antony , Ooi Jenny , Chang Lisa , Khurana Ishant , Olson Eric N , El-Osta Assam , Porrello Enzo R
Published: July 2021

Epigenetic modifications have emerged as central players in the coordination of gene expression networks during cardiac development. Much attention has focused on the role of histone modifications during embryonic heart development, but relatively little is known about the epigenetic control mechanisms that guide post-natal heart maturation. Furthermore, few studies have investigated the role of DNA methylation during cardiac development, despite the fundamental importance of this biological process for transcriptional regulation. The purpose of the current study was to determine whether DNA methylation plays an important role in guiding transcription during the neonatal period, which is an important developmental window for cardiac maturation, including cardiomyocyte cell cycle withdrawal and loss of endogenous regenerative capacity. Here, we interrogated genome-wide changes in transcription and CpG methylation during post-natal cardiac maturation in the mouse (P1 vs. P14). CpG sequencing (CpG-seq) identified 2251 differentially methylated regions (DMRs) between P1 and P14. Intersection of DMRs with different genomic features revealed that 1248 DMRs were associated with promoter regions and transcription start sites. Interestingly, increased methylation of genes associated with well-known signalling pathways for muscle development and differentiation, such as the bone morphogenetic protein, fibroblast growth factor, Wnt and Notch signalling pathways, was associated with transcriptional repression of these regulatory networks at P14. To determine the functional significance of these dynamic changes in the cardiac methylome, we inhibited DNA methylation in vivo by administration of the DNA hypomethylating agent 5-azacytidine from P2 to P12. Post-natal inhibition of DNA methylation caused a marked increase in heart size and was associated with increased cardiomyocyte proliferation. This study provides evidence for widespread alterations in DNA methylation during post-natal heart maturation and suggests that DNA methylation may play an important role in the transcriptional silencing of key regulatory networks for muscle development and cardiomyocyte proliferation during neonatal life.
Abstract 122: A Novel Mutation in a X-linked Gene Causes Human Congenital Dilated Cardiomyopathy
Authors: Chun Young Wook , Feaster Tromondae K , Williams Charles H , Sheng Calvin C , Frist Audrey Y , Su Yan Ru , Bichell David P , Hong Charles C
Published: July 2021

Congenital dilated cardiomyopathy (cDCM) is a rare but often fatal disease. In most cases, there is no family history, and its etiology is unknown. A major hurdle to elucidating a mechanistic understanding of congenital cardiomyopathy, and primary cardiomyopathies in general, has been a lack of access to diseased human cardiac tissues. Recent advances in patient-derived induced pluripotent stem cells (iPSCs) now enable production of human cardiomyocytes (iPSC-CMs) and allows for a systematic study of normal and diseased cardiomyocytes. We hypothesize that cardiomyocytes generated from iPSCs derived from cDCM patients will exhibit cellular and molecular differences from those generated from healthy donor iPSCs and that a rare genetic mutation, or a collection of mutations, plays a critical role in cDCM pathogenesis. To test these hypotheses, we generated cardiomyocytes from iPSCs derived from a 7-month old male with cDCM using a robust cardiac induction protocol based on the “matrigel sandwich” method of Kamp and colleagues. With this remarkably robust induction method, iPSC-CMs from the cDCM patient and a healthy control donor exhibited proteomic profiles that were 99.7% superimposable. Despite the close similarity at the global proteome level, iPSC-CMs from the cDCM patient showed greatly reduced contractility and dramatic structural defects in the sarcomere and the mitochondria. Finally, bioinformatics analyses of the RNAseq data of the patient’s iPSC-CMs discovered a putative causal mutation in an evolutionarily conserved site in a X-linked gene with unknown function. In summary, our work demonstrates that iPSC-based approaches are particularly useful for the study of human congenital heart diseases. We plan to confirm the causality of this mutation using gene editing techniques such as CRISPR/Cas9 and explore the role of this novel gene in cardiomyocyte structure and function.
Abstract 123: Optical Mapping of Host and Human Embryonic Stem Cell-Derived Cardiomyocyte Graft Electrical Activity in Injured Hearts
Authors: Filice Dominic , Zhu Wei-Zhong , Van Biber Benjamin , Hauch Kip D , Laflamme Michael A
Published: July 2021

Human embryonic stem cell-derived cardiomyocytes (hESC-CMs) show tremendous promise for cardiac repair, but more information is required as to their electrical behavior in vivo. hESC-CMs expressing the protein calcium sensor GCaMP3 provide a graft autonomous reporter of activation, which we have used to show hESC-CM grafts can couple with host myocardium in injured hearts. When we sought to assess host-graft electrical interactions by optical voltage mapping, we found the commonly used lipophilic voltage dyes RH237 and di-4-ANEPPS label host but not graft tissue. We hypothesized the water-soluble voltage dye di-2-ANEPEQ could overcome this limitation and efficiently label both host and graft. After confirming good spectral separation of GCaMP3 and di-2-ANEPEQ signals by spectrofluorimetry and confocal spectral imaging, we transplanted 1x10^8 GCaMP3+ hESC-CMs into guinea pig hearts (n=6) at 4 weeks following cardiac injury and then imaged engrafted hearts at 2 weeks post-transplantation with a dual-channel CCD-based system. We found a differential time-course of di-2-ANEPEQ labeling between host and graft tissues, with stable optical action potentials (APs) obtained in host and graft tissue after ~4 and ~13 minutes of dye perfusion, respectively. This differential labeling kinetics, which was also observed on washout, presumably reflects sluggish graft perfusion and provides another tool for distinguishing host and graft signals. No regions of 1:1 host-graft coupling were identified, and graft tissue had spontaneous rates from 0.1-2 Hz and long optical AP durations from 500-1090 ms. Activation maps based on di-2-ANEPEQ and GCaMP3 signals indicated relatively slow conduction velocities in graft tissue, as well as patterns of propagation that commonly occurred along a vector distinct from that in host tissue. These imaging studies reveal multiple potentially pro-arrhythmic properties in hESC-CM graft tissue including slow propagation, ultralong AP duration, as well as aberrant patterns of activation that can vary from beat to beat. We conclude the electrical behavior of both graft and host myocardium can be reliably assessed by the simultaneous imaging of a graft-autonomous fluorescent reporter of activation and a water-soluble voltage dye.
Abstract 130: Ox-LDL Impairs The Survival Of Bone Marrow Stem Cells Partially Through Membrane Damage Independent Of ROS Production In Vitro
Authors: Li Xin , Xiao Yuan , Cui Yuqi , Zhu Hua , Narasimhulu Chandrakala A , Zhang Jia , Hao Hong , Liu Lingjuan , Xie Xiaoyun , He Guanglong , Ma Jianjie , Verfaillie Catherine M , Parthasarathy Sampath , Liu Zhenguo
Published: July 2021

Aims:
cell-based therapy with bone marrow stem cells (MSCs) remains a viable option for tissue repair and regeneration. One of the major challenges for cell-based therapy is the limited survival of the cells after in vivo administration. The exact mechanism(s) for impaired in vivo survival of the implanted MSCs remains to be defined. Oxidized low-density lipid protein (ox-LDL) is a natural product in human blood, and the major contributor to the development of atherosclerosis. The present study was to investigate the effect of ox-LDL on the survival of bone marrow stem cells and the mechanisms in vitro.

Methods and Results:
Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL (with the final concentration of 10 and 20 ug/ml) for up to 48 hours. Exposure to ox-LDL resulted in significant cell death and apoptosis of MAPCs in association with a significant increase in LDH release in the conditioned media in a dose- and time-dependent manner, indicating significant cell membrane damage. The membrane damage was further confirmed with the rapid entry of the small fluorescent dye FM1-43 as detected using confocal microscope. Ox-LDL generated a significant amount of reactive oxygen species (ROS) in the culture system as measured with electron paramagnetic resonance spectroscopy. The antioxidant N-acetylcysteine (NAC, 0.1 mM) completely inhibited the production of ROS from ox-LDL. However, it didn’t prevent ox-LDL-induced cell death or apoptosis. However, pre-treatment of the cells with the specific membrane protective recombinant human MG53 protein (rhMG53)(66 ug/ml, final concentration) significantly, reduced LDH release and the entry of FM1-43 dye into the cells exposed to ox-LDL.

Conclusion:
Ox-LDL enhanced cell death and apoptosis of MAPCs with a mechanism independent of ROS generation in vitro. Ox-LDL impaired the survival of MAPCs partially through cell membrane damage in vitro.