bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 32: Intravenous Mobilized Autologous Peripheral Blood CD34+ Stem Cell Transplantation for Angina
Authors: Lim Hadyanto , Dharma Lindarto , Umar Zein , Ilham Hariaji
Published: June 2021

Background:
Intramyocardial CD34+ stem cell therapy for patients with refractory angina shows that this is safe and feasible. We aimed to determine whether intravenous transplantation of mobilized autologous peripheral blood CD34+ stem cells provides beneficial effects for patients with angina pectoris.

Methods:
We administered granulocyte colony stimulating factor (G-CSF, 5.0 μg/kg/day) subcutaneously once a day for 4 days to 15 patients (4 women and 11 men aged 50-78 years) with intractable angina pectoris (Canadian Cardiovascular Society functional class III-IV) for mobilization of CD34+ cells into the peripheral blood. Ischemia was assessed by exercise stress testing. Leukapheresis procedure was started on the day 4 of G-CSF using the Spectra Optia cell separator. Circulating and intravenous transplantation of autologous CD34+ cells after leukapheresis were measured by flow cytometry. The effects of G-CSF on blood were measured by hematology analyzer and semi-auto chemistry analyzer.

Results:
Intravenous peripheral blood CD34+ cells increased after leukapheresis (from 1.12±0.48 cells/μL to 107.42±23.83 cells/μL, p<0.001) and total white blood cells count (from 7.82 ± 2.63x10
3
/μl to 37.47±15.07 x10
3
/μl, p<0.001). Indices of hsCRP, platelets, hemoglobin, alanine aminotransferase, lactic dehydrogenase, and uric acid were not changed by treatment. At week 4, angina frequency was significantly lower after intravenous CD34+ cells (from 15.07±4.03 to 3.27±1.49, p<0.001). Similarly, improvement in exercise tolerance was significantly higher by stem cell transplantation (from 5.90±2.53 minutes to 8.41±2.49 minutes, p<0.001). Most patients reported mild myalgia which were easily managed with acetaminophen.

Conclusions:
Intravenous autologous CD34+ stem cell transplantation improved angina frequency and exercise tolerance. The cell mobilization and leukapheresis procedures were found safe and tolerable in patients with angina pectoris.
Abstract 321: miR-125b-3p Cox20 Axis Deregulation in the Novel Combined Model of Pulmonary Fibrosis, Pulmonary Hypertension
Authors: Ruffenach Gregoire , Umar Soban , Eghbali Mansoureh
Published: June 2021

Introduction:
Pulmonary fibrosis (PF) is a life threatening disease characterized by lung scaring. In some cases PF is complicated by pulmonary hypertension (PH). Growing evidence demonstrates the role of oxidative stress and the implication of micro-RNAs in PF and PH but their exact role in PF associated with PH (PH-PF) remain unclear. Recently, our group developed a combined model of PF-PH in rats and demonstrated a robust up-regulation of miR125 in rats with PF-PH compared to PF. Interestingly, the cytochrome C oxidase assembly factor 20 (COX20), a protein implicated in respiratory chain complex IV structure and oxidative stress regulation, is predicted to be a target of miR125. Based on these data, we hypothesis that: upregulation of miR125 in the lungs of combined PF-PH lead to COX20 down-regulation and therefore increases oxidative stress.

Methods:
Wistar male rats were divided to 5 groups. The rats received either a single intra-tracheal instillation of bleomycin (2.5mg.kg
-1
, PF group) or PBS in day 0 (control). Two weeks later some of the rats in PF group received MCT injection (60mg.kg
-1
, PF-PH group). All the rats were sacrificed at day 35 (n=4 rats per groups). Disease severity at day 35 was assessed by direct heart catheterization and Masson trichrome staining. Real-time PCR was used to quantify expression of COX20 and gluthation peroxidase 1 (GPX1), an anti-oxidant protein.

Results:
PF-PH rats developed a more severe PH than PF alone (58.02±4.3 vs. 37.59±5.9mmHg,). Histological analysis revealed an increase in pulmonary fibrosis and vascular remodelling in lung of PH-PF rats compare to PF alone. Mechanistically we found COX20 was robustly downregulated in PF-PH compared to PF group (~200 fold vs. ~10 fold, in relative to control, p<0.05) This drastic decrease in COX20 in PF-PH expression was concomitant with an increase in oxidative stress highlighted by reduced expression of GPX1 in PF-PH compared to PF alone (~20000 fold vs. ~400 fold, relative to control).

Conclusion:
Our results implicate miR-125/COX20 axis in PH-PF model and pave the way to a better understanding of PH-PF disease mechanism
Abstract 34: Human Cardiac Mesenchymal Cell Derived Extracellular Vesicles Promote Angiogenesis Through Angiopoietin-TIE2 Signaling
Authors: Pathan Asif , Farid Talha , Khan Abdur Rahman , Nasr Marjan , Wysoczynski Marcin , Bolli Roberto
Published: June 2021

Cell-based therapy is considered a promising approach to treat the damaged heart due to myocardial infarction. Although the mechanisms for their beneficial action are not yet clear, exosome/extracellular vesicles (EVs) secreted by these cells may be involved in their reparative paracrine signaling. Previous studies have suggested that EVs isolated from several cell types (e.g. cardiosphere-derived cells, embryonic stem cell, CD34+ stem cells) induce angiogenic activity both
in vitro
and
in vivo
. Here, we investigated whether EVs secreted by adult human cardiac mesenchymal cells (hCMCs) exhibit pro-angiogenic activity, and if so, what signaling molecules are involved in this process. hCMCs were isolated from right atrial appendage of patients undergoing cardiac procedures and were characterized by the expression of classical mesenchymal markers- CD29 (99.1%), CD73 (99.0%), CD90 (20.4%), CD105 (99.3%), CD 31 (16.8%), CD34 (0.9%) and CD45 (0.1%). EVs isolated from serum-free 24-hour hCMC conditioned media using PEG4000-based precipitation technique exhibited two distinct population of particles with size range of 10-60nm and 100-500nm in diameter; expressed characteristic exosomal markers- CD63, HSP70, Flotillin-1 and were negative for cellular organelle markers- calreticulin (ER and apoptotic bodies), prohibitin (mitochondria), GM130 (Golgi), Lamin B (nuclear protein), β-actin (cytoskeleton) and PMP70 (peroxisomes) as determined by immunoblotting. In vitro assays revealed that hCMC EVs promote human umbilical cord endothelial cells (HUVECs) proliferation, transwell migration in Boyden chamber and tube formation on Matrigel, indicative of enhanced angiogenesis. Angiogenic proteomic array identified that angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) proteins are highly enriched in EVs secreted by hCMCs. Furthermore, hCMC EV mediated HUVEC migration and tube formation was inhibited by TIE2 kinase inhibitor. Overall, these findings suggest that ANG-1 and ANG-2 are the key component of hCMC secreted EVs and they promote angiogenesis by activating TIE2 receptor in endothelial cells.
Abstract 350: Chromosome Content of Cardiac Progenitor Cells Influence on Regenerative Potential in the Heart
Authors: Broughton Kathleen , Khieu Tiffany , Nguyen Nicky , Mohsin Sadia , Quijada Pearl , Wang Jessica , Monsanto Megan , Gude Natalie , Sussman Mark
Published: June 2021

Discovery of endogenous stem cells found within the heart, cardiac progenitor cells (CPC), has prompted intense basic discovery in multiple experimental animal models and clinical trials in heart failure patients. A survey of the literature reveals animal or cellular models exhibiting regenerative properties are also characterized by genome duplication or polyploidization. Our lab recently discovered a fundamental difference between large and small mammalian CPCs through karyotype analysis: rodent (rat, mouse) CPCs possess mononuclear tetraploid (4n) chromosome content, whereas large animal (human, swine) CPCs are mononuclear diploid (2n). This primary distinction between large and small animals prompts provocative questions regarding regenerative potential as well as the translational applicability of regenerative studies performed in rodent models. To expand on this finding, CPCs were isolated from eight human tissue samples (normal and heart failure), two swine strains (Gottingen and Yorkshire) six mice strains (FVB, C57, CAST, SPRET, SAMP6, and SAMR1), and seven rat clonal lines (Sprague Dawley), which confirmed karyotyped ploidy content per species as characterized through flow cytometry and confocal microscopy techniques. The large (human, swine) and small (rat, mouse) mammal CPCs maintained stable chromosome content through increased passaging and positive correlations appears between chromosome content and growth rate through increased passages as well as chromosome content and unrestrained growth through increased passages. The ploidy content of endogenous CPCs was investigated
in situ
with heart tissue sections with comparable ploidy determinations to that of cultured CPCs. Furthermore, the unique tetraploid content of murine CPCs is reinforced by comparison with ckit+ progenitor cells of secondary tissues (intestine and bone marrow) and cardiomyocytes
in situ
and
in vitro
that exhibit mostly diploid (2n) chromosome content per nuclei. These studies suggest ploidy may play a significant role in the biological capacity of the CPC and the endogenous stem cell pool may influence the regenerative capacity of the heart. Future directions will focus on defining the advantage of polyploid content in mediating regeneration.
Abstract 352: The Effect of the Extracellular Matrix of Failing Hearts on Cardiac Stem Cells
Authors: Delfín Dawn A , DeAguero Joshua , McKown Elizabeth N
Published: June 2021

Objective:
Stem cell therapy to repair cardiac damage, caused by the pathological remodeling that occurs in cardiovascular disease, represents a cutting edge therapeutic option toward preventing and treating heart failure. There is a paucity of information on how the phenotypes of stem cells are altered in the “hostile” environment of pathologically remodeled hearts. The objective of this study was to (a) determine the phenotypes of cardiac stem cells cultured on failing versus normal human heart tissue, and (b) to characterize pathology of failing human heart samples.

Methods:
We isolated the extracellular matrix (ECM) from end-stage failing human hearts (compared to control human hearts) and used the isolated ECM to coat tissue culture plates. The ECM represents the scaffold of the heart in which stem cells will integrate, proliferate, and differentiate. We then cultured induced pluripotent stem cell-derived cardiac progenitor cells on the cardiac-ECM plates and analyzed their phenotypes (morphology, differentiation into cardiomyocytes, and gene expression). We also performed analysis of the failing cardiac tissue itself to determine the extent of fibrosis and expression of various ECM and matricellular proteins.

Results:
Cells cultured on failing heart ECM showed important differences compared to those on control heart ECM, including reduced cell-matrix adhesion and altered responses to beta-adrenergic stimulation. The failing heart shows increased fibrosis and differential expression of specific ECM and matricellular proteins.

Significance:
We will use these data to determine how best to direct cardiac stem cells used for tissue generation in severely damaged hearts toward differentiation into functioning cardiomyocytes.
Abstract 356: Sam68 Promotes NF-kB Signaling and Inflammation in Macrophages and Impedes the Recovery of Arterial Injury
Authors: Han Shuling , Zhou Junlan , Qin Gangjian
Published: June 2021

Background:
The role of Src-associated-in-mitosis-of-68-kDa (Sam68) protein in vascular biology has not been studied. A recent report suggests that Sam68 suppresses TNF-a-mediated NF-kB activation in embryonic fibroblasts. Since NF-kB plays a critical role in vascular inflammation and injury, we sought to dissect the molecular mechanism by which Sam68 regulates NF-kB signaling and its functional significance in vascular injury.

Methods & Results:
The endothelial denudation injury was induced in the carotid arteries of Sam68
-/-
mice and WT littermates. Sam68
-/-
mice displayed a significantly accelerated re-endothelialization and attenuated neointimal hyperplasia, which was associated with reduced macrophage infiltration and lowered expression of pro-inflammatory cytokines (i.e., TNF-a, MCP-1, IL-1, and IL-6) in the injured vessels. The improved carotid recovery in Sam68
-/-
mice was recapitulated in WT mice that had received Sam68
-/-
bone-marrow (BM) transplantation, suggesting that Sam68 impedes vascular recovery primarily by its function in BM cells, likely pro-inflammatory cells. In cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNF-a-induced expression of pro-inflammatory cytokines and in the level of P-IKKαβ, P-IkBa (in cytosol) and P-P65 (in nucleus), indicating attenuated NF-kB activation. These results were confirmed in peritoneal and BM-derived macrophages from Sam68
-/-
and WT mice. Furthermore, Sam68 co-immunoprecipitation and mass-spectrometric analyses identified cytoskeleton protein Filamin A (FLNA) as a novel Sam68-interacting protein in response to TNF-a stimulation. Reverse co-immunoprecipitation and truncational mutagenesis confirmed that Sam68-FLNA interactions require the N-terminus of Sam68. Finally, analyses in Raw264.7 cells with FLNA knockdown revealed that the effects of Sam68 on TNF-a[[Unsupported Character - Codename ­]]-induced NF-kB signaling and pro-inflammatory cytokine expression were dependent on FLNA.

Conclusions:
Our data suggest that Sam68 promotes pro-inflammatory response in injured arteries and impedes recovery, and this effect may be attributable, at least partially, to the exaggerated NF-kB activity in macrophages.
Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis
Authors: Liu Jie , Qi Yanmei , Hsu Shu-Chan , Saadat Siavash , Rahimi Saum , Lee Leonard , Yan Chenghui , Tian Xiaoxiang , Han Yaling , Li Shaohua
Published: June 2021

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.
Abstract 363: In Situ Cardiac Cellular Reprogramming Using Adenoviral Vectors: Implication for Clinical Myocardial Regeneration
Authors: Mathison Megumi , Singh Vivek P , Chiuchiolo Maria J , Sanagasetti Deepthi , Mao Yun , Patel Vivekkumar B , Yang Jianchang , Kaminsky Stephen M , Crystal Ronald G , Rosengart Todd K
Published: June 2021

Objective:
The reprogramming of cardiac fibroblasts into induced cardiomyocytes (iCMs) improves ventricular function in myocardial infarction models. Only integrating chronic expression vectors have thus far been used to administer reprogramming genes, potentially limiting clinical applicability. We hypothesized that reprogramming could be achieved using non-integrating, acute expression adenoviral vectors.

Methods:
Adenoviral (Ad) and lentiviral vectors encoding Gata4 (G), Mef2c (M) and Tbx5 (T) were validated
in vitro
. Sprague Dawley rats then underwent coronary artery ligation and Ad-mediated administration of vascular endothelial growth factor to provide infarct prevascularization. Three weeks later, Ad or lentivirus encoding G, M, or T or an equivalent dose of a null vector was administered. Outcomes were analyzed by serial echocardiography, and by terminal MRI and histology.

Results:
Ad and lentivirus vectors provided equivalent in vitro GMT expression by Western blotting and AdGMT induced expression of the cardiomyocyte marker cTnT in approximately 7% of cardiac fibroblasts, compared to 4% of cells infected with LentiGMT. Sections of infarcted myocardium from rats that had been treated with AdGMT or LentiGMT demonstrated higher density of cells expressing the cardiomyocyte marker MHY7 compared to AdNull treated animals (p<0.05). Echocardiography demonstrated that AdGMT significantly increased ejection fraction compared to AdNull (AdGMT: 21% ± 3%, LentiGMT: 14% ± 5%, AdNull: -0.4% ± 2%; p<0.05).

Conclusions:
Adenoviral vectors are at least as effective as lentiviral vectors in inducing cardiac fibroblast transdifferentiation into iCMs and improving cardiac function in post-infarct rat hearts. The utility of short-term expression Ad vectors represents an important potential tool in inducing cardiac cellular reprogramming clinically.
Abstract 366: Cardiac Strain From Magnetic Resonance Imaging Offers Sensitive Detection of Therapeutic Efficacy Following Transplantation of Tissue Engineered Heart Muscles
Authors: Qin Xulei , Riegler Johannes , Tiburcy Malte , Denney Thomas , Zimmermann Wolfram , Wu Joseph
Published: June 2021

Background:
Myocardial infarction (MI) leads to gradually decreased cardiac contractility that can ultimately cause heart failure or sudden death. One potential therapy to recover this MI-impaired contractile function is the regenerative approach that integrates both tissue engineering and exogenously produced cardiomyocytes. Although this contractile recovery has been assessed by different imaging methods in previous studies, these assessments in global cardiac performance are plagued by their relatively low sensitivity. As this treatment is locally performed, the sensitivity of its therapeutic assessment can be improved by myocardial strain imaging that assesses regional contractility.

Methods:
We first made tissue engineered heart muscles (EHMs) by casting human embryonic stem cell-derived cardiomyocytes (ESC-CMs) with collagen in preformed molds. Then we transplanted these EHMs (n=12) into the post-MI rat hearts one month after this injure. We also performed sham surgeries on a control group (n=9) without EHM transplantation. Meanwhile, strain imaging from cardiac magnetic resonance imaging (MRI) and ultrasound were performed prior to and four weeks after EHM transplantations. In addition, global cardiac performance such as left ventricular ejection fraction (EF) was estimated from cardiac MRI.

Results:
Based on the measurements, regional myocardial strain measured from tagged MRI detected the preserved systolic function in the EHM-treated group compared to control group (P=0.04). In contrary, the global function EF was unable to detect this significant difference between both groups (P=0.38). Similarly, myocardial strain measured from cardiac ultrasound also failed to detect the significance between both treatment and control groups (P=0.24).

Conclusions:
This study for the first time confirmed that regional assessment of contractile function offered higher sensitivity compared to the conventional measure of global systolic performance. This finding highlights the feasibility and sensitivity of cardiac strain derived from tagged MRI as a sensitive method to assess the efficacy of regenerative therapies in both preclinical and clinical studies.
Abstract 370: Hedgehog Signaling Regulates Cardiac Regeneration
in vivo
Authors: Singh Bhairab N , Gong Wuming , Garry Mary G , Koyano-Nakagawa Naoko , Garry Daniel J
Published: June 2021

The adult mammalian heart has a limited regenerative capacity due primarily to reduced cardiomyocyte (CM) proliferation. Here, we demonstrated hedgehog (HH) signaling pathway as an essential regulator of heart regeneration and CM proliferation. We undertook genome-wide screening using a novel algorithm,
bootstrap,
which showed an induction of HH signals in the regenerating newt heart. Blockade of HH signaling in the resected newt heart resulted in complete ablation of cardiac regeneration and scar formation. EdU-labeling revealed that inhibition of the HH pathway significantly reduced CM proliferation by 3-fold (n=4 at each time period post-injury). In mammals, cardiac specific loss- and gain-of-function of HH signals demonstrated its role in CM proliferation and regeneration in the postnatal heart. Genetic deletion of
floxed-Smoothened
(

Smo
L/L

) allele at postnatal day 2 (P2) inhibited neonatal heart regeneration with impaired cardiac function and scarring following injury. Conversely, induction of constitutively active Smoothened (SmoM2) at P7 stimulated CM proliferation by 2.5-fold (n=3) and regeneration after myocardial infarction during the non-regenerative window. Lineage-tracing experiments showed that activation of
Smo
contributed to heart regeneration by promoting proliferation of the pre-existing cardiomyocytes. Activation of HH signals in the cultured CM at P1 and P7 showed an increased proliferative response by 2- and 3-fold (n=4; 1900 cells evaluated for each condition), respectively. Mechanistically, ChIP-seq analysis revealed that HH signals promoted the proliferative program by directly regulating the expression of cyclin-dependent kinases including cyclinD2, cyclinE1 and Cdc7. Finally, activation of HH signaling in the terminally differentiated hiPSC-derived CM resulted in an increase in the number of α-Actinin
+
/EdU
+
and α-Actinin
+
/Ki67
+
cells by 2.5-fold (n=3; 645 cells assessed for each condition) and 3-fold (n=3; 685 cells assessed for each condition), respectively. These studies defined an evolutionarily conserved function of HH signaling from newt to mouse to human, as a key regulator of cardiomyocyte proliferation and regeneration that may serve as a platform for regenerative therapies.
Abstract 374: FGF2 Isoform-selective as well as Non-selective Signals Protect Cardiomyocytes from Doxorubicin-induced Cell Death
Authors: Koleini Navid , Nickel Barbara E , Fandrich Robert R , Kirshenbaum Lorrie A , Cattini Peter A , Kardami Elissavet
Published: June 2021

Introduction:
Doxorubicin (DOX) induced cardiotoxicity, including cardiomyocyte cell death, remains a major challenge for anticancer therapies. DOX can disrupt the balance between synthesis and degradation systems by affecting major cellular signaling pathways including the mammalian target of Rapamycin (mTOR), and AMP-activated kinase (AMPK). Fibroblast Growth Factor 2 (FGF2), a multifunctional protein expressed by heart cells and downregulated by DOX, consists of two types of isoforms, low molecular weight, Lo-FGF2, (~18 kDa) and high molecular weight, Hi-FGF2, (>20 kDa), displaying isoform-selective and non-selective properties. We have studied the effects of added FGF2 isoforms on DOX induced dysregulation of signaling pathways impacting on cardiomyocyte survival and autophagy.

Methods and Results:
Primary cultures of neonatal rat cardiomyocytes were subjected to 0.5 μM DOX in the absence or presence of pretreatment with FGF2 isoforms, at 10 ng/ml. Both Hi- and Lo- FGF2 decreased DOX-induced injury and cell death, measured by LDH release, a Live-Dead assay, and activation of caspase 3. Neither isoform prevented the DOX-induced cell death in MCF7 cells, a breast cancer cell line. In myocytes, both isoforms prevented the DOX-induced downregulation of mTORC1 activity (phospho-Ser2448). The mTOR inhibitor Rapamycin prevented the beneficial effects of FGF2. Hi-FGF2, but not Lo-FGF2, significantly increased AMPK activity (phospho-Thr172) pre- and post-DOX. Compound C, an inhibitor of AMPK, prevented the protective effect of Hi- , but not Lo-, FGF2. To assess autophagy, cardiomyocytes were transduced with an adenoviral vector for GFP-LC3. Numbers of GFP-LC3 aggregates (dots) per cell were measured, and suggested that both Hi- and Lo-FGF2 prevented the DOX-induced increases in autophagy and autophagy flux, as also indicated by increased levels of p62 post-DOX in the presence of FGF2. Pharmacological inhibition of autophagy by 3-Methyladenine was protective against DOX.

Conclusions:
FGF2 isoforms protect cardiomyocytes against acute DOX damage, by activating mTORC1 (both Hi or Lo FGF2), as well as, in the case of Hi-FGF2, the AMPK pathway. Protection by FGF2 isoforms was associated with prevention of accelerated flux.
Abstract 378: Cholecystokinin Upregulates Cholesterol Absorption in Human Intestinal Epithelial Cells
Authors: Guo Zhongmao , Zhang Ningya , Dent Lemuel , Okoro Emmanuel U , Yang Hong
Published: June 2021

Excessive absorption of intestinal cholesterol is a risk factor for atherosclerosis. We previously reported that cholecystokinin (CCK) increases intestinal cholesterol absorption and plasma cholesterol level in mouse models. The goal of this study was to investigate the effect of CCK on cholesterol absorption and Niemann-Pick C1 Like 1 (NPC1L1) expression in human primary intestinal epithelial cells (HPIECs). Normal HPIECs were isolated from small bowl resection specimens, and purchased from the Lonza Group (Walkersville, MD)
.
Cholesterol absorption was determined by measuring transcellular cholesterol transport in adherent cell culture and cholesterol association and release in suspension cell culture. Surface NPC1L1 was isolated using a biotinylation kit and detected by western blotting. Our data demonstrate that HPIECs express both CCK receptor-1 and -2 (CCK1R; CCK2R). Treatment of HPIECs with 3 nM [Thr28, Nle31]-CCK for 60 min increased transcellular cholesterol transport, cholesterol association and release by ~38, 32 and 44%. Selective inhibition of CCK1R and CCK2R with antagonists (1 μM lorglumide or L365260) or selective knockdown of CCK1R and CCK2R with siRNAs attenuated CCK-induced cholesterol absorption. In the cells cultured on transwell membranes, CCK increased the level of NPC1L1 in the apical membrane by ~35% but did not alter the total NPC1L1 protein expression. Inhibition or knockdown of NPC1L1 attenuated CCK-induced cholesterol absorption. These data imply that activation of CCK1R/2R enhances cholesterol absorption by induction of NPC1L membrane translocation. [This study was supported by NIH grants U54MD0007593, UL1TR000445, and SC1HL101431]
Abstract 38: Engineered Anisotropic Scaffolds Promote the Function of Cocultured Cardiomyocytes Derived From Human Pluripotent Stem Cells
Authors: Wanjare Maureen , Jung-Woong Kim Joseph , Huang Ngan
Published: June 2021

Since the heart is effectively an anisotropic organ in which the cardiomyocytes (CM) are locally aligned in series, it is important to engineer cardiac tissues that promote CM alignment in order to closely mimic the architecture of the native tissue, as well as better mimic the cellular composition of the heart. The objective of this study was to define the role of anisotropic extracellular matrix cues on the organization and survival of human induced pluripotent stem cell-derived CMs (hiPSC-CMs) by co-culturing hiPSC-CMs and primary endothelial cells (ECs) on parallel-aligned microfibrillar scaffolds. The hiPSC-CMs were generated from hiPSCs using small molecule Wnt pathway agonists and antagonists. Subsequently, the hiPSC-CMs were sequentially seeded on day 15 after EC attachment. We cultured monocultures and cocultures on electrospun three-dimensional (3D) scaffolds of polycaprolactone (PCL) and polyethylene oxide (PEO) polymer blends with an average fiber diameter of 14 μm. Aligned scaffolds were fabricated by stretching the randomly oriented scaffolds by 300% of the original scaffold length. Randomly oriented fibrillar scaffolds had an average pore diameter of 17 μm when compared to the 36 μm pore diameter of aligned scaffolds. Our results indicate that alignment of co-cultured cells at a 5:1 hiPSC-CMs : EC ratio was promoted by anistropic 3D electrospun scaffolds when compared to similar random 3D electrospun scaffolds. Additionally, cocultured cells on aligned fibrillar scaffolds had a mean angle of orientation of 30.8°, relative to the direction of fibrils, which was similar to that of hiPSC-CM monocultures on aligned scaffolds (32.8°). In contrast, the degree of alignment of hiPSC-CMs on randomly oriented fibrillary scaffolds was 43.4°, which suggests a non-oriented population of cells. Aligned scaffolds also produced more synchronized cardiomyocyte contraction than random scaffold orientations, although both induced spontaneous contraction frequency of ~1Hz. This study highlights the importance of nanotopographical cues and intercellular interactions in mediating the morphology and contractility of hiPSC-CMs for treatment of cardiovascular diseases such as myocardial infarction.
Abstract 382: Cell Type-specific Fibroblast Growth Factor Receptors That are Regulated in Human Heart Disease
Authors: Peter Angela K , Bugg Darrian N , Ozeroff Christopher D , Chonchol Michel , Ambardekar Amrut V , Kakkar Rahul , Leinwand Leslie A
Published: June 2021

Chronic kidney disease (CKD) is characterized by increased serum levels of the phosphaturic hormone fibroblast growth factor 23 (FGF23), the development of left ventricular hypertrophy, and increased risk of mortality due to cardiovascular disease. The FGF receptors (FGFRs) responsible for facilitating the effect of FGF23 on the heart and the expression patterns of FGFRs in the different cell types in the heart remain largely uncharacterized. We hypothesized that cardiomyocytes express a different FGFR profile than cardiac fibroblasts and that this differential expression profile could contribute to cell type-specific responses to FGF23. We measured FGFR mRNA levels at baseline and following pro-hypertrophic growth factor stimulation in isolated adult rat ventricular cardiomyocytes (ARVMs), neonatal rat ventricular cardiomyocytes (NRVMs), and neonatal rat ventricular fibroblasts (NRVFs). At baseline, NRVMs expressed predominantly FGFR1 but also expressed FGFRs 2 and 3. In ARVMs, FGFRs 1 and 3 predominated while FGFRs 2 and 4 were expressed at very low levels. FGFRs in ARVMs and NRVMs were unchanged by growthfactor stimulation. At baseline, NRVFs expressed equal amounts of FGFRs 1, 2 and 3 which were all downregulated with FGF23 stimulation.

To determine whether these results translated to human hearts, we examined the FGFR profile in a cohort of non-failing, hypertrophic cardiomyopathy (HCM), and ischemic cardiomyopathy (ICM) human patient samples. Compared to non-failing patients, we found FGFR2 was significantly increased in HCM patients and trended towards an increase in ICM patients. The cardiac FGFR2 profile matched the FGF23 profile. In addition, we found that FGFR4 was significantly down regulated in both ICM and HCM compared to non-failing patients.

In conclusion, different cell types in the heart differ in their FGFR profiles at baseline. The FGFR profile does not change in NRVMs or ARVMs in response to FGF23 treatment, but the expression of FGFRs 1, 2, and 3 decrease in NRVFs in response to FGF23. HCM and ICM in humans is characterized by an increase in FGFR2 and FGF23 expression accompanied with a downregulation of FGFR4 expression. Modulating FGFRs in the myocardium may provide a novel therapeutic target in CKD patients.
Abstract 384: Evaluating Late Sodium Current in Human Induced Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Schocken Derek , Stohlman Jayna , Zhang Xiaoyu , Abassi Yama , Johannesen Lars , Vicente Jose , Strauss David , Blinova Ksenia
Published: June 2021

Background:
Inhibiting late sodium current (I
NaL
) reduced drug-induced QTc prolongation in a recent clinical trial. Induced pluripotent stem cell derived cardiomyocytes (iPSC-CMs) have emerged as a valuable tool in preclinical assessment of multichannel blocking drugs’ potential to prolong QT and induce arrhythmias. However, sodium channels in commercially available iPSC-CMs are known to be under expressed, necessitating investigation into the presence and effects of I
NaL
in this electrophysiological model.

Methods and Results:
A platform combining simultaneous measurements of field potential and contraction (xCELLigence RTCA CardioECR, ACEA Biosciences) was used to assess the acute effects of three I
NaL
enhancing drugs, ATX-II, ibutilide, and alfuzosin, given alone or in combination with an I
NaL
blocker, lidocaine in iPSC-CMs (iCell Cardiomyocytes
2
, Cellular Dynamics). Additionally, dofetilide, diltiazem, and lidocaine alone were included as positive controls for hERG, L-type calcium, and sodium channel block.

ATX-II, a potent and specific I
NaL
enhancer, caused significant dose dependent rate-corrected field potential duration (FPDc) prolongation, which was then subsequently reduced in a dose dependent manner by the addition of lidocaine. At 100 nM ATX-II prolonged the FPDc by 1153.8 ± 135.8 ms from 360.5 ± 16.4 ms at the baseline, which was then reduced to 537 ± 37.4 ms with the addition of 30 μM lidocaine. Ibutilide (0.1-1 μM), a class III antiarrhythmic, caused beating rate decreases and early after depolarizations (EADs) that were not affected by lidocaine addition. Alfuzosin, which increases both peak and late sodium currents, caused dose-dependent reduction of beating rate, FPDc prolongation, and EADs at 5 μM and 10 μM. Alfuzosin-induced EADs were mitigated by addition of lidocaine (5-15 μM).

Conclusions:
Late sodium current enhancers prolonged repolarization and induced arrhythmias in human iPSC-CMs. These effects were reversed by addition of lidocaine, a specific late sodium current blocker. These results are consistent with the late sodium current being present in iPSC-CMs in the presence of a late sodium current enhancer, which may have implications for drug safety testing.
Abstract 385: Broccoli Sprout Extracts Prevents Diabetic Cardiomyopathy in
db/db
Mice: The Importance of Up-regulated Nrf2 Expression and Function
Authors: xu Zheng , Tan Yi , Sun Jian , Cai Lu
Published: June 2021

This study was to define whether consumption of broccoli sprout extracts (BSE) can be used as natural sulforaphane (SFN) supplement to systemically up-regulate NFE2-related factor 2 (Nrf2) for the prevention of diabetic cardiomyopathy (DCM). Five-months-old db/db and age-matched wild type (WT) mice were fed BSE or SFN by gavage every other day for 3 months. Both db/db and WT mice were divided into four groups: vehicle (0.1 ml/10g), BSE-low dose (delivery SFN 0.5 mg/kg), BSE-high dose (delivery SFN 1.0 mg/kg), and SFN (0.5 mg/kg) as a positive control. After 3 months treatment, cardiac function was evaluated by echocardiography. Cardiac hypertrophy, fibrosis, inflammation, and oxidative stress damage were assessed by Western blot, real-time PCR, and histopathological examination. Both BSE and SFN significantly prevented diabetes-induced cardiac dysfunction, hypertrophy, and fibrosis (increased accumulation of collagen and expression of connective tissue growth factor). BSE and SFN also prevented diabetes-induced cardiac inflammation and oxidative injury. Like SFN, the positive control of Nrf2 activator, BSE significantly up-regulated Nrf2 expression and transcription activity, reflected by increased Nrf2 nuclear accumulation and the expression of its downstream genes at both mRNA and protein levels. These results indicated that the effect of BSE-high dose is similar to those of SFN, suggesting that BSE can be used as SFN nature supplement to up-regulate Nrf2 expression and function for the prevention of DCM in type 2 diabetes. Since BSE is a health supplement, the potential for its clinical application for diabetic patients is highly promised.
Abstract 386: Notch1 Signaling Negatively Modulates Repolarizing Kv Currents in Cardiomyocytes
Authors: Borghetti Giulia , Signore Sergio , Sorrentino Andrea , Goichberg Polina , Rota Marcello
Published: June 2021

Notch1 is a critical signaling pathway during embryonic and early postnatal development and activity of this transduction system gradually declines after birth. Conversely, outward K
+
Kv currents are progressively increased in myocytes during postnatal life leading to shortening of the action potential (AP) duration and acquisition of the mature electrical phenotype. Thus, we tested the possibility that Notch1 signaling modulates the electrophysiological properties of developing cells by interfering with Kv current densities. For this purpose, molecular and physiological studies were conducted in vitro using mouse neonatal myocytes (NMs). These assays were complemented with tests in adult cells obtained from a conditional mouse model of Notch1 intracellular domain (NICD) overexpression. NMs were treated with a γ-secretase inhibitor (DAPT) to prevent the cleavage of Notch1 receptor and subsequent translocation of the active NICD to the nucleus. By RT-PCR, Notch1 targets Hes1 and Hey1 were reduced with DAPT-treatment, confirming the effective perturbation of Notch1 signaling. Importantly, inhibition of Notch signaling led to upregulation of the three splice variants of the Kv channel-interacting proteins 2 (KChIP2) gene, which controls the appearance of outward Kv currents in myocytes. By patch-clamp, Kv currents were identified in only 15% of NMs (n=20) cultured for 1-2 days in in control condition, whereas this fraction increased to 50% (n=18) in cells treated with DAPT. Thus, Notch inhibition promotes the expression of KChIP2 and appearance of Kv currents. To clarify these in vitro findings, the consequences of ectopic activation of Notch1 signaling on the electrical behavior of adult myocytes were established. By patch-clamp, NICD-overexpressing myocytes presented a >2-fold longer duration of the early repolarization phase of the AP, with respect to cells from wild type hearts. This alteration was coupled with a >50% reduction of Kv currents I
to
and I
Kslow1
in NICD-overexpressing cells, with respect to wild-type myocytes. Thus, Notch signaling represses KChIP2 and Kv currents in cardiomyocytes representing an important modulator of the electrical phenotype of the developing heart.
Abstract 388: Functional Role of Inositol 1,4,5-Trisphosphate Receptors in Cardiac Hypertrophy
Authors: Garcia Martha Iveth
Published: June 2021

Calcium is a second messenger that is essential for the control of a variety of cellular functions. In the heart, calcium plays an integral role in many cellular processes including energy metabolism, contraction, gene expression, and cell death. The inositol 1,4,5-trisphosphate receptor (IP
3
R) is a calcium channel that is ubiquitously expressed throughout the body. There are three IP
3
R isoforms encoded by separate genes. In the heart, the IP
3
R-2 isoform is reported to being most predominant with regards to both expression levels and functional significance. Several groups have shown that perinuclear IP
3
R-2 may play a key role in the progression of hypertrophy by mediating nuclear calcium release in response to endothelin-1 (ET-1) and angiotensin II. However, the functional roles of IP
3
R-1 and IP
3
R-3 in the heart are essentially unexplored despite measureable expression levels. We hypothesize that all three IP
3
R isoforms can contribute to cytosolic calcium transients and cardiac stress induced by ET-1. As our model we used dissociated neonatal and adult rat ventricular cardiomyocytes. We found readily detectable levels of expression of all three IP
3
Rs in both neonatal and adult rat ventricular cardiomyocytes. We found that in addition to the perinuclear area, all three IP
3
R isoforms were expressed throughout the cardiomyocyte including the sarcoplasmic reticulum. Using isoform specific siRNA, we found that knocking down individual IP
3
Rs did not alter ET-1 induced calcium responses in neonatal cardiomyocytes. However, knocking down all three isoforms abolished the effects of ET-1 on calcium homeostasis. We next tested whether IP
3
Rs mediate nuclear-specific calcium elevations. Using a genetically encoded calcium indicator targeted to the nucleus we were able to specifically discriminate nuclear versus cytosolic calcium transients after ET-1 treatment. Contrary to previous reports, we found no evidence that IP
3
R-mediated calcium release is confined to the nucleus. Our findings that IP
3
R isoforms are functionally redundant in the heart have significant implications for animal models of hypertrophy and human disease.
Abstract 389: Amino Terminal Region of Cardiac Myosin Binding Protein-C is Necessary for Cardiac Function
Authors: Lynch Thomas L , Kuster Diederik W , Sivaguru Mayandi , Previs Michael J , Lee Kyounghwan , Govindan Suresh , Craig Roger , Warshaw David M , Sadayappan Sakthivel
Published: June 2021

Rationale:
Cardiac myosin binding protein-C (cMyBP-C) is a thick filament-associated protein that has been suggested to regulate cardiac contraction via its amino terminal (N’) region. However, the necessity of the N’-C0-C1f region (domains C0 through C1 and the first 17 residues of the M-domain) of cMyBP-C in regulating cardiac function
in vivo
has not been elucidated.

Hypothesis:
The N’-C0-C1f region of cMyBP-C is critical for normal cardiac function
in vivo
.

Methods and Results:
Transgenic mice with 80±4% expression of a truncated cMyBP-C missing the N’-C0-C1f region (cMyBP-C
110kDa
) were generated and characterized at 3-months of age. cMyBP-C
110kDa
animals exhibited cardiac hypertrophy as suggested by an increased heart weight to body weight ratio (5.0±0.1 mg/g NTG vs. 6.9±0.1 mg/g cMyBP-C
110kDa
, p<0.0001) and an elevation in pathological hypertrophy markers determined by real-time PCR. Histopathological analysis showed increased cardiac fibrosis in cMyBP-C
110kDa
hearts compared to hearts from non-transgenic (NTG) littermates. Intriguingly, increased phosphorylation of cMyBP-C at Ser-282 and Ser-302, sites important for cMyBP-C’s regulation of actomyosin interactions, was observed in cMyBP-C
110kDa
hearts compared to controls. Electron microscopy revealed normal sarcomere structure in cMyBP-C
110kDa
hearts but with apparently weaker cMyBP-C stripes. Furthermore, the ability of cMyBP-C to slow actin-filament sliding within the C-zone of native thick filaments isolated from NTG hearts was lost on thick filaments from cMyBP-C
110kDa
hearts. Short-axis M-mode echocardiography indicated a significant elevation in left ventricular internal diameter and a significant reduction in fractional shortening (31±5% NTG vs. 16±3% cMyBP-C
110kDa
, p=0.0003) in cMyBP-C
110kDa
hearts compared to controls. Finally, global longitudinal strain analysis revealed abnormal wall motion in cMyBP-C
110kDa
hearts. Based upon these data, we propose that the N’-region of cMyBP-C is a critical regulator of actomyosin interactions and controls aberrant contraction kinetics within the cardiac sarcomere.

Conclusion:
The N’-C0-C1f region of cMyBP-C regulates cardiac contractility and is necessary for maintaining normal cardiac function
in vivo
.
Abstract 392: Decreased ARNT Mediates Cardiovascular Endothelial Dysfunction in Diabetic Cardiomyopathy
Authors: Knapp Maura , Zheng Mei , Huang Hao , Liao James K , Wu Rongxue
Published: June 2021

Background:
Endothelial dysfunction is thought to be one of the key risk factors leading to cardiac dysfunction. We have previously shown that ARNT is a critical regulator of cardiac metabolism and its deletion in the heart mimics diabetic cardiomyopathy. Here, we hypothesize that reduced ARNT expression in the endothelium may lead to endothelial dysfunction and contribute to diabetic cardiomyopathy.

Methods and Results:
Primary cardiac endothelial cells (mCVEC) were isolated from DB/DB mouse hearts and confirmed using vWF staining and flow cytometry. Isolated mCVEC from DB/DB mice showed more than a 50% reduction in ARNT protein levels, suggesting that endothelial ARNT may play a role in diabetic hearts. We generated a mouse with an endothelial specific ARNT deletion (ecARNT
-/-
) by crossing ARNT flox/flox mice with Cre recombinase mice under the control of the VE-Cadherin promoter. Deletion of ARNT in the endothelium was achieved by the administration of oral tamoxifen chow. ecARNT
-/-
mice displayed cardiac hypertrophy and worsened cardiac function after high-fat chow feeding. In vitro studies were done using siRNA technology to knockdown ARNT in mCVEC. Knockdown of ARNT did not increase cell viability at base level, but led to impaired capillary-like endothelial tube formation and reduced cell migration in response to high glucose treatment. Nitric oxide (NO) production (a marker of endothelial dysfunction) and eNOS expression were also reduced after ARNT knockdown. To determine the underlying mechanisms by which ARNT may regulate endothelial metabolism we performed a DNA microarray and confirmed our results using RT-PCR. We discovered a significant induction of NF-kB and its target genes, including ELAM-1 and ICAM-1. These changes are similar to those we observed in mCVEC from DB/DB mouse hearts. Taken together, this data shows that a reduction in ARNT may regulate endothelial dysfunction in the diabetic heart through an inflammatory pathway.

Conclusion:
Endothelial ARNT may be a critical mediator of endothelial function and could serve as a therapeutic target for diabetic cardiomyopathy.
Abstract 394: Multiple Cellular Mechanisms Underlie the Trafficking-deficient Phenotype for Kv11.1 (hERG) Mutations Linked to Long QT Syndrome
Authors: Smith Jennifer L , Reloj Allison R , Anderson Corey L , January Craig T , Delisle Brian T
Published: June 2021

Type 2 Long QT syndrome (LQT2) is commonly caused by missense mutations that disrupt the trafficking of Kv11.1 channels. The goal of this study is to determine the cellular mechanisms that underlie the trafficking-deficient phenotype for LQT2-linked mutations located in different channel domains. We expressed the pore-domain mutation G601S and the cyclic nucleotide binding domain mutation F805C in human embryonic kidney 293 (HEK293) cells or human inducible pluripotent stem cell derived cardiomyocytes (hiPSC-CMs). HEK293 cells or hiPSC-CMs expressing G601S showed a diffuse intracellular anti-Kv11.1 staining pattern that colocalized with the transitional endoplasmic reticulum (ER) marker BAP31, but not markers for the rough ER (calnexin), ER-associated degradation compartment (derlin1), ER exit sites (Sec31), or the ER-Golgi intermediate compartment (ERGIC53). Culturing cells in the drug E-4031 (10 μM, 24 hrs), which corrects the G601s trafficking-deficient phenotype, decreased its colocalization with BAP31. Unlike G601S, the trafficking-deficient phenotype for F805C is not corrected by culturing cells in E-4031. Expressing F805C in HEK293 cells or hiPSC-CMs showed a very different anti-Kv11.1 staining pattern that consisted of several discrete aggregates. In hiPSC-CMs, the F805C aggregates partially overlapped with derlin1 staining and co-transfected green fluorescent protein (gfp) in the cytosol. Incubating cells in the proteasome inhibitor bortezomib (10 μM, 4 hrs) dispersed the F805C aggregates to generate a diffuse intracellular staining pattern. We conclude that G601S and F805C localize to distinct ER subcompartments: G601S is sequestered in the transitional ER and F805C localizes to an ER associated degradation subcompartment that is sensitive to proteasome inhibition.