bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 239: p53 Protects Cardiac Stem Cells From the Toxic Effects of Chemotherapy and Controls their Fate
Authors: Kannappan Ramaswamy , Palano Giorgia , Goichberg Polina , Sanada Fumihiro , Signore Sergio , Kim Junghyun , Rota Marcello , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

Doxorubicin (DOXO) causes dilated cardiomyopathy and heart failure. We have documented previously that DOXO-mediated cardiotoxicity is dictated by functional alterations of cardiac stem cells (CSCs). DOXO-induced myopathy was coupled with a reduction in CSC number due to increased death, inhibition of proliferation, and senescence. We raised the possibility that survival and growth of CSCs following DOXO treatment may be enhanced by modulating the intracellular level of p53, which plays a critical role in the determination of stem cell fate. For this purpose, transgenic mice carrying an additional p53 allele (Sp53) were studied. With respect to wild-type mice (WT), CSCs isolated from Sp53 mice (Sp53-CSCs) showed increased apoptosis with accumulation of the pro-apoptotic p53 targets BAX, PUMA and Pidd. Conversely, the expression of p21Cip1, a cell cycle inhibitor and inducer of cell senescence, was lower in Sp53-CSCs than WT cells. Upon DOXO treatment, Sp53-CSCs exhibited accelerated onset of apoptosis. However, viable Sp53-CSCs showed enhanced formation of DNA damage response foci, indicative of a very efficient DNA repair mechanism. Following removal of DOXO, Sp53-CSCs re-entered the cell cycle and divided, while WT cells continued to die by apoptosis or became senescent. The response of WT-CSCs to DOXO involved the pro-apoptotic Bcl2 family member Noxa and the senescence-associated protein p16INK4a. In contrast, exposure of Sp53-CSCs to DOXO provoked pulses of p53 expression, which favored sustained upregulation of Mdm2. Mdm2 antagonized the inhibitory effect of p53 on cell growth and prevented apoptosis. Ultimately, Sp53-CSCs showed accumulation of PCNA, which is required for DNA repair and synthesis. Importantly, IGF-1 release was higher in Sp53-CSCs, promoting their replication through an autocrine-paracrine mechanism. Collectively, our data demonstrate that changes in the pattern of p53 expression have beneficial effects on CSCs by amplifying the DNA repair response, facilitating the clearance of cells with non-repairable DNA damage, and enabling the proliferation of cells in which DNA lesions are effectively removed. Thus, targeting p53 expression in CSCs may protect the heart from the toxic effects of chemotherapy.
Abstract 241: In Vivo Response to Dynamic Hyaluronic Acid Hydrogels
Authors: Young Jennifer L , Tuler Jeremy , Braden Rebecca , Schup-Magoffin Pamela , Schaefer Jacquelyn , Kretchmer Kyle , Christman Karen L , Engler Adam J
Published: July 2021

Tissue-specific elasticity arises in part from developmental changes in extracellular matrix over time, e.g. ~ 10-fold myocardial stiffening in the chicken embryo. When this time-dependent stiffening is mimicked in vitro with a thiolated hyaluronic acid (HA-SH)/poly(ethylene glycol) diacrylate (PEGDA) hydrogel, improved cardiomyocyte maturation has been observed. However, host interactions, matrix polymerization, and stiffening kinetics remain uncertain in vivo, and each plays a critical role in therapeutic applications using HA-SH. In order to assess in vivo feasibility and biocompatibility of HA-SH/PEGDA hydrogels, subcutaneous injections were first performed. Hematological and histological analysis of subcutaneously injected HA-SH/PEGDA hydrogels showed minimal systemic immune response and host cell infiltration. Most importantly, subcutaneously injected HA-SH/PEGDA hydrogels exhibited time dependent porosity and stiffness changes at a rate similar to hydrogels polymerized in vitro, as measured by atomic force microscopy. When injected intramyocardially, host cells begin to actively degrade HA-SH/PEGDA hydrogels within 1-week post-injection, continuing this process while producing matrix to nearly replace the hydrogel within 1 month post-injection. While non-thiolated HA did not degrade after injection into the myocardium, it also did not elicit an immune response, unlike HA-SH/PEGDA, HA-SH/low concentration PEGDA, or high concentration HA-SH only hydrogels, where visible granulomas and macrophage infiltration were present at 1 month post-injection, as indicated by CD45 (lymphocyte marker) and CD68 (macrophage marker) staining, likely due to reactive thiol groups. Altogether, these data suggest that the HA-SH/PEGDA hydrogel responds appropriately in a less vascularized niche and stiffens as had been demonstrated in vitro, but in more vascularized tissues, in vivo applicability appears limited.
Abstract 243: End Stage heart Failure Cardiac Stem Cells has Strong Regenerative Ability compared to Congenital Heart Cardiac Stem cells
Authors: Mishra Rachana , Sharma Sudhish , Simpson David , Desmukh Savitha , Kaushal Sunjay
Published: July 2021

Cellular based therapy is an important component of cardiac regenerative medicine. We have already identified and characterized a small population of undifferentiated cells present throughout the human heart that have the characteristics of cardiac stem cells. To date, no study has examined the potential application of human cardiac derived stem cells (hCDCs) generated from End Stage Heart Failure (ESFH) pediatric patient’s heart. In this study, we characterized samples from ESFH (recipients of heart transplant) and CDH (donors of the heart transplantation) to quantify the expression of various cardiac progenitor markers by immunofluorescence, flow cytometry and RT-PCR. The numbers of cardiac progenitor cells were highest in the right atria as compared to other chambers of the end-stage heart. FACS and IF analysis demonstrated significantly higher c-kit (9.36±1.384 vs 3.056±0.39, n=5) and ISL1 (33.61±4.0 vs 3.708±0.93) expressions in hCDCs derived from ESFH compare to CDH myocardium. Also, there was a tendency for increased FLK1 and Sca-1 expression in ESFH patients respectively. To determine, the functional potential, hCDCs were transplanted into a rodent myocardial infarct (MI) model. ESFH-derived hCDCs produced a stronger recovery of ventricular function than CDH-derived hCDCs (ESFH, n=5, EF=57+3% vs. CDH, n=5, 41.5±3%, P<0.05). We also observed significantly higher secretion of VEGF (ESFH, n=5: 1.928±0.3623 vs CDH, n=5: 0.93±0.08, P<0.05) and SDF-1α (ESFH, n=5: 3.1±0.6934 vs CDH, n=5: 0.99±0.1471, P<0.05) by the ESFH-derived hCDCs as compared to CDH-derived hCDCs. This correlated with increased angiogenesis in the MI model at 28days. This study demonstrates that ESFH-derived hCDCs are highly functional and secrete angiogenic cytokines, more than CDH-derived hCDCs. To understand the mechanism of this activity will be critical for future clinical study.
Abstract 250: Adult Cardiac Stem Cells regulation by microRNAs
Authors: Matsuda Alex J , Pinheiro Ana I , Amaral Andreia , Gama-Carvalho Margarida , Rosário Luís , Kajstura Jan , Anversa Piero
Published: July 2021

In recent years stem and progenitor cells have been identified in the heart, changing our understanding and therapeutic approach of heart disease. These cells can proliferate and differentiate in vitro into beating cardiomyocytes, endothelial and vascular smooth muscle cells, providing the potential for tissue regeneration and repair.

microRNAs (miRs) have been identified as master switches controlling proliferation and differentiation, and in particular as key regulators of stem cells and cardiac development and function. Modulation of miR-regulated gene expression networks holds the potential to control cell fate and proliferation, with predictable biotechnological and therapeutic applications.

We have characterized the expression profile of a subset of 100 miRs with reported functions in stem cell and tissue differentiation in mouse Sca1+ cardiac progenitor cells (CPCs). CPC miR expression profiles were compared with bone marrow mesenchymal stem cells (MSCs) and mouse embryonic heart (E=9) to obtain insights into the origins and function of this rare cell population. Within the miR panel that was studied, a subset with relative high expression levels in CPCs was identified, which may act as negative regulators of differentiation and proliferation. Although the overall expression profile of Sca1+ CPCs is closer to BM-MSCs, the most highly expressed miRs in CPCs are distinctive and predicted to target key genes involved in the control of cell proliferation and adhesion, vascular function and cardiomyocyte differentiation.

Our results provide novel insights into the gene expression networks active in CPCs, which will now be explored in functional studies to identify key regulators of proliferation and differentiation.
Abstract 253: Modeling of Pathologic Hypertophy with Cardiac Microtissues
Authors: Alvarez Rosa M , Margulies Kenneth B
Published: July 2021

Introduction:
Pathological myocyte hypertrophy and fibroblast activation are key features of myocardial remodeling in hypertension and heart failure. Crosstalk between myocytes and fibroblasts contributes to the structural, mechanical, and electrical remodeling involved in various forms of heart failure. Replicating these responses in traditional 2D cell culture conditions has proven difficult. In this study, we utilized a novel platform to examine the cellular crosstalk and biological processes that underlie functional and structural changes in the 3D engineered tissues when exposed to pathological factors. We hypothesized that engineered cardiac tissues can report structural and functional responses to pathologic biochemical stimuli.

Methods:
These studies employ microfabricated polydimethylsiloxane (PDMS) templates to engineer arrays of 3D cardiac microtissues (CMT). Cantilevers within templates provide physiologically-relevant biomechanical loading to the CMTs, promote the appropriate organization of neonatal rat cardiac myocytes and fibroblasts, and report active and passive force generation in real time. Data was collected using fluorescent and confocal microscopy.

Results:
CMTs in a controlled mechanical environment demonstrate mechanical and structural changes within 24 hours of exposure to a cocktail of factors involved in maladaptive remodeling, including Transforming Growth Factor-β, Endothelin-1, and Angiotensin II. Analysis of 40 microtissues revealed that the percent change in passive tension was 45% higher (16.4 ± 10.3% vs. 61.1 ± 11.8%, p<0.01) and in active tension 170% higher (24.9 ± 9.5% vs.196.1 ± 76.0%, p<0.001) in treated CMTs compared to untreated CMTs. The percentage change in cross-sectional area of CMTs treated with pathological factors was 35% higher (-24.5 ± 15.6% vs.11.6 ± 7.9%, p<0.001) than untreated samples.

Conclusions:
These findings demonstrate that the CMT model can be used to study and dissect the dynamics of pathological hypertrophy. Ongoing experiments are examining the distinct contributions of the pathologic growth factors, the relative contributions of fibroblasts and cardiomyocytes to the pathologic growth response, and the interactions between biomechanical and humoral inputs.
Abstract 254: Defects in c-kit Receptor Function Affect Myocyte Turnover Resulting in Premature Myocardial Aging in the W/WV Mouse
Authors: Caballero Maria-Virginia , Signore Sergio , Kannappan Ramaswamy , Kim Junghyun , Ogórek Barbara , Wybieralska Ewa , Borghetti Giulia , Valeriani Fiorenza , Cannata Antonio , Graciotti Laura , Leri Annarosa , Rota Marcello , Kajstura Jan , Anversa Piero
Published: July 2021

The objective of this study was to determine whether attenuation of cardiac stem cell (CSC) growth in the adult heart interferes with physiologic cell turnover leading to premature accumulation of senescent myocytes and myocardial aging. For this purpose, W/W
V
mice, which carry a mutated non-functional c-kit receptor, were studied. The W/W
V
mouse is a compound heterozygous mutant mouse. The W mutation corresponds to a large amino acid deletion that includes the transmembrane domain of the c-kit receptor while the W
V
mutation is a point mutation in the kinase domain of this receptor. These genetic modifications reduce c-kit kinase activity by approximately 80-90% affecting hematopoiesis, gametogenesis and melanogenesis. Echocardiographically, at 2-3 months of age, LV diameters and ejection fraction were similar in controls and W/W
V
mice. However, at 5-6 months and 8-9 months, diastolic and systolic LVDs were larger in W/W
V
mice than in WT. Ejection fraction decreased with age in W/W
V
mice. These various aspects of negative ventricular remodeling typically occur with the development of the aging myopathy in mice 24 month-old and older. Moreover, ventricular dilation is commonly associated with wall thinning, both critical parameters of diastolic and systolic wall stress. Importantly, these anatomical and functional changes in W/W
V
mice were associated with a marked increase in the percentage of CSCs and myocytes expressing the senescence-associated marker p16
INK4a
. At two months of age, wild-type mice and W/W
V
mice had the same number of LV myocytes which were essentially identical in volume. However, in comparison with wild-type mice, W/W
V
mice at 4 months of age had a decreased number of LV myocytes which were larger in volume. The magnitude of myocyte proliferation at the time of sacrifice was assessed by two markers of the cell cycle, Ki67 and MCM5. On average, cycling myocytes were 3-4-fold higher in wild type mice than in W/W
V
mice. In contrast, apoptotic myocytes, evaluated by the TdT assay and hairpin 1 in situ ligation, were significantly more frequent in W/W
V
mice than in WT mice. In conclusion, mutation of the c-kit receptor in CSCs impairs new myocyte formation and promotes accumulation of senescent myocytes.
Abstract 255: Modulation Of Cardiac Macrophages As A Strategy For Infarct Healing
Authors: Somasuntharam Inthirai , Carroll Sheridan , Brown Milton , Garcia Andres , Davis Michael
Published: July 2021

Heart failure is the leading cause of death in the developed world and myocardial infarction (MI) is the most common cause. Macrophages are key cells that orchestrate the initial inflammatory as well as later stage wound healing responses following MI. These functions are carried out by pro-inflammatory (M1) and reparative (M2) macrophages respectively. Optimal healing response after MI requires a balancing act of the biphasic macrophage response, so as to not prolong inflammatory signals detrimental to wound healing. Taking advantage of the fact that interleukin-4 (IL-4) activates macrophages towards M2, we hypothesize that delivering IL-4 to the post-MI heart can alter the ratio of M2 to M1 macrophages in the infarct area and induce a better healing response. In this study, we validate our approach in vitro and perform in vitro optimization of a suitable delivery system.

RAW 264.7 macrophages were stimulated with IL-4 (10ng/uL) or LPS/IFN-γ (100ng/mL and 10ng/mL) for 24h and gene expression markers (qPCR) and Nitric Oxide (NO) levels (Griess assay) analyzed as indication of M1or M2 activation. Mouse aortic endothelial cells were treated with conditioned media from these cells for 24h and tube formation assessed on matrigel. A bioactive, protease-cleavable polyethylene glycol (PEG) hydrogel delivery system was evaluated for release of functional IL-4 to LPS-activated macrophages. Empty or IL-4 encapsulating hydrogel was placed on a trans-well above LPS-stimulated macrophages. Collagenase I at 0.1mg/mL was applied over 48h to degrade the gels and release IL-4 (n≥3 and p<0.05 considered significant by one-way ANOVA).

We demonstrate that IL-4 significantly upregulates M2 markers (MRC-1 and Arg-1) while IFN-γ and LPS upregulate M1 markers (NO and TNF-alpha). We observe enhanced tube density in endothelial cells treated with M2 media while M1 inhibited tube formation. Hydrogel release study shows a significant reduction in NO levels of LPS-stimulated macrophages when IL-4 is released, demonstrating that IL-4 is released from the gel in its bioactive form. In conclusion, we show that macrophages can indeed respond to changing stimuli and adopt distinct activation types and our PEG based hydrogel could be a potential delivery system for in vivo IL-4 delivery.
Abstract 257: Bone Marrow Cells Form New Cardiomyocytes in the Infarcted Myocardium
Authors: Sanada Fumihiro , Kim Junghyung , Czarna Anna , Signore Sergio , Sorrentino Andrea , Mangiaracina Chiara , Kannappan Ramaswamy , Isobe Kazuya , Goichberg Polina , Rota Marcello , Hosoda Toru , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

The ability of c-kit-positive bone marrow cells (c-kit-BMCs) to form cardiomyocytes following delivery to the acutely infarcted myocardium is a matter of debate. In an attempt to resolve the controversy, we raised the possibility that c-kit-BMCs represent a functionally heterogeneous pool, containing cells with distinct transdifferentiation potential. To test this hypothesis, c-kit-BMCs were infected with EGFP-lentiviruses and injected in infarcted hearts. At 1 and 2 weeks, the regenerated myocardium was enzymatically digested and EGFP-labeled myocytes, endothelial cells (ECs), fibroblasts, and c-kit-cells were sorted. By employing a PCR-based method of detection of viral integrants, we searched for unique sites of viral insertion in the infected c-kit-BMCs and their progeny. Common insertion sites were found in the DNA of all cell populations, documenting that single c-kit-BMCs transdifferentiated into multiple cell lineages. However, the 31 detected clones shared 9 distinct sites of integration, suggesting that myocytes in different animals were generated by specific subsets of c-kit-BMCs, which retained the ability to transdifferentiate. To strengthen the observation that myocardial regeneration is oligoclonal in nature, freshly isolated FACS-sorted c-kit-BMCs were simultaneously transduced with 3 lentiviruses, each encoding red, green or blue (RGB) fluorescent proteins. Different combinations of inserted vectors resulted in the formation of numerous c-kit-BMC clones of mixed colors. RGB-infected HSCs were injected in infarcted rats; at 4 days, engrafted c-kit-BMCs showed a polyclonal pattern characterized by the presence of cells labeled by most of the color combinations seen in vitro. However, at 2 weeks, only a few of the homogeneously colored colonies seen at 4 days persisted and formed large patches of cells composed of myocytes, ECs, and smooth muscle cells. Our findings document that c-kit-BMCs are functionally heterogeneous and have different ability to survive, engraft, and transdifferentiate in the ischemic heart. At present, clonal c-kit-BMCs are being analyzed by RNA-sequencing for the identification of the unique phenotypical properties of c-kit-BMCs with high transdifferentiation potential.
Abstract 258: Hypoxic and Normoxic Niches Regulate Cardiac Stem Cell Function
Authors: Sanada Fumihiro , Kim Junghyung , Czarna Anna , Chan Noel Y , Signore Sargio , Isobe Kazuya , Sorrentino Andrea , Mangiaracina Chiara , Wybieralska Ewa , Cimini Maria , Borghetti Giulia , Rota Marcello , Hosoda Toru , Kajstura Jan , Anversa Piero , Leri Annarosa
Published: July 2021

In bone marrow niches, hypoxia favors stem cell quiescence and normoxia is required for stem cell proliferation and commitment. The objectives of this work were to determine whether
a)
differences in O
2
content regulate the function of cardiac stem cells (CSCs); and
b)
defects in tissue oxygenation with aging affects niche homeostasis. At 3 and 30 months of age, hypoxic and normoxic CSCs were found in the myocardium, but the senescent mouse heart was characterized by a 1.5-fold increase in hypoxic CSCs. At both ages, hypoxic CSCs were quiescent and lineage negative while cycling and early committed cells were restricted to the normoxic pool. Importantly, telomeres were longer in hypoxic than normoxic CSCs and this difference was more apparent in old mice. Telomere length did not change with age in hypoxic CSCs but decreased 40% in normoxic CSCs. As a consequence, at 3 and 30 months, the fraction of normoxic CSCs expressing the senescence-associated marker p16
INK4a
was, respectively, 6-fold and 2.7-fold higher than that of hypoxic CSCs. Collectively, these findings indicate that the old heart contains a larger fraction of CSCs, which are forced in a quiescent state and do not participate in myocyte turnover. To define whether hypoxic CSCs can be activated, senescent mice were kept in an atmosphere of 70% O
2
to increase O
2
content in the hypoxic niches. Hyperoxia decreased significantly the fraction of hypoxic CSCs at 1 day and this response persisted at one week, possibly favoring myocyte formation. These in vivo results suggest that O
2
levels in CSCs may be regulated by the capillary network and the degree of tissue oxygenation. The distance between CSCs and the closest capillary was significantly higher for hypoxic than for normoxic CSCs. The numerical density of capillaries per mm
2
of myocardium computed within a radius of 30 μm from hypoxic CSCs decreased markedly with age. Thus, diffusion distance for O
2
to CSCs appears to constitute a major determinant in the maintenance of the hypoxic state of CSCs. Collectively, our data demonstrate that a balance between hypoxic and normoxic niches is present in the young heart but is disrupted later in life when capillary rarefaction expands the pool of hypoxic quiescent CSCs, which are no longer involved in myocyte replacement.
Abstract 259: Role of Connexin 43 in Human Bone Marrow Derived Mesenchymal Stem Cell Cardiac Integration and Cardiac Stem cell Niche Formation.
Authors: Sanina Cristina , Rodrigues Claudia , Bellio Michael , Schulman Ivonne , Balkan Wayne , Hatzistergos Konstantinos , Margitich Irene , Hare Joshua
Published: July 2021

Introduction:
Bone marrow-derived mesenchymal stem cells (MSCs) and cardiac progenitor cells (CPCs) have been used successfully as a cell-based approach for cardiac regeneration after myocardial infarction. While MSCs and CPCs are effective, in part due to differentiation, paracrine effect, and integration into the heart, the mechanism underlying these effects remains controversial.

Objective:
We hypothesized that functional connexin 43 (Cx43) gap junctions are crucial for MSC and CPCs interaction and integration into cardiac tissue and cardiac stem cell niche formation.

Methods and Results:
Human MSCs were co-cultured with neonatal rat ventricular cardiomyocytes (NRVMs). The ability of MSCs to form gap junctions was modulated using lentiviral constructs to either knockdown (Cx43KD) or overexpress (Cx43OE) Cx43. Co-culture of Cx43OE or control MSCs with NRVMs led to the formation of beating, three-dimensional tubes whereas Cx43KD MSCs failed to form tubes (n=5, p<0.05). Furthermore, we replicated the cardiac stem cell niche by combining human CPCs and MSCs in an in vitro model (the same lentiviral constructs were used). As a result, Cx43 expressing MSCs and CPCs formed organotypic three-dimensional structures similar to human MSCs and NRVMs, whereas the lack of Cx43 significantly affected the structure formation (n=6, p<0.05). Cx43KD significantly increased proliferation (n=5, p<0.05) and changed culture phenotype (n=4, p<0.05) of MSCs or CPCs cultured separately. The angiogenic tube length, assessed by a Matrigel assay in vitro, was significantly reduced in MSCs and HUVEC Cx43 KD groups compared to control (n=5, p<0.05). The change/onset of endothelial (KDR, PECAM-1, VE-cadherin), and cardiomyocyte (Nkx2.5, Gata4, Troponin I, Na+ channel, K+ channel, Ca2+ channel, Na+-Ca2+ exchanger) gene expression was investigated in co-culture of MSCs and CPCs. Only voltage ion channel gene expression was upregulated in control and Cx43OE groups by co-culture (n=5, p<0.05), but not affected in Cx43KD group.

Conclusion:
These findings reveal that cell−cell contact mediated by Cx43 gap junctions enables MSCs to interact with CPCs, integrate and reconstitute cardiac stem cell niches.
Abstract 267: Opposing Functions of Two Splice Variants of the Human Transcription Factor Grainyhead-like 3 in Endothelial Cells And in vivo
Authors: Altschmied Joachim , Büchner Nicole , Jakob Sascha , Farrokh Sabrina , Goy Christine , Schaal Heiner , Haendeler Judith
Published: July 2021

Grainyhead-like 3 (GRHL3) is a member of the evolutionary conserved Grainyhead family of transcription factors. In humans, three isoforms are derived from differential first exon usage and alternative splicing, which differ only in their N-terminus. Isoform 2, the only variant also present in mouse, is required for endothelial cell (EC) migration and protects against apoptosis. The functions of the human specific isoforms 1 and 3, which are derived from an alternatively spliced pre-mRNA, have not yet been investigated, although all three isoforms are expressed in EC. Therefore, we have assessed their effects on EC migration and apoptosis.

Overexpression of the two proteins had opposite effects on EC migration, with isoform 1 acting pro-migratory. This protein also protected EC against apoptosis in an eNOS-dependent manner, whereas isoform 3 had no effect. These opposing outcomes with respect to apoptosis EC were corroborated by isoform-specific knockdowns.

With reporter assays using a GRHL3-specific luciferase reporter we demonstrated that both are active transcription factors. Microarray analyses revealed that they induce divergent target gene sets in EC. Two validated targets, Akt2 and Mxi1, which are upregulated by isoform1, are regulators of Akt1-, and thus eNOS-phosphorylation and apoptosis, which could explain the effects of this protein on these processes.

In vivo, overexpression of isoform 3 in zebrafish embryos resulted in increased lethality and severe deformations, while isoform 1 had no deleterious effect.

In conclusion, our data demonstrate that the splice variant derived isoforms 1 and 3 of the human transcription factor GRHL3 induce opposing effects in primary human endothelial cells and in a whole animal model, most likely through the induction of different target genes.
Abstract 269: Cardiac Specific Disruption of Drp-1 Causes the Development of Cardiac Dysfunction via Inhibition of Autophagy and Induction of Apoptosis
Authors: Ikeda Yoshiyuki , Sadoshima Junichi
Published: July 2021

Fission and fusion affect mitochondrial turnover in part by modulating mitophagy. This study aimed to clarify the role of mitochondrial fission in regulating cardiac function and autophagy in the heart. Dynamin-related protein 1 (Drp-1) plays an essential role in mediating mitochondrial fission. Therefore, we generated cardiac specific Drp-1 KO mice and utilized cultured cardiomyocytes transduced with adenovirus harboring short hairpin Drp-1 (Ad-shDrp-1) to test the effect of Drp-1 disruption both in vivo and in vitro. In Drp-1 KO hearts we observed a significantly greater mitochondrial mass ratio compared to control, as assessed by electron microscopy (Drp-1 KO: 3.57 ± 1.38, control: 1.18 ± 0.31, P<0.05). Mitochondrial ATP content was significantly lower (0.70 ± 0.07 vs 1.03 ± 0.10, P<0.05), while mitochondrial swelling was significantly greater (% decrease in absorbance; 8.01 ± 1.99 vs 2.01 ± 0.58, P<0.05) in Drp-1 KO hearts versus control. Mitochondrial membrane potential, assessed by JC-1 staining, was significantly reduced in myocytes with knockdown of Drp-1. Taken together, these results suggest that inhibition of fission causes mitochondrial dysfunction. We also examined the effect of Drp-1 depletion on autophagy. We found that the amount of LC-3 II was significantly less (0.47 ± 0.16 vs 1.32 ±0.34, P<0.05), whereas p62 expression was significantly greater (1.14 ± 0.16 vs 0.16 ± 0.06, P<0.01) in Drp-1 KO hearts compared to control. The number of LC3 dots in Ad-shDrp-1 transduced myocytes was lower than that of sh-scramble treatment. We investigated apoptosis and found that the amount of cleaved caspase-3 (0.62 ± 0.24 vs 0.18 ± 0.04, P<0.05) and the number of TUNEL positive cells (0.22 ± 0.12 vs 0.03 ± 0.06%, P<0.01) were higher in Drp-1 KO versus control hearts. Cardiac systolic function was reduced (ejection fraction; 44.5 ± 6.3 vs 85.4 ± 5.7%, P<0.01) and LVW/tibia length was greater (4.48 ± 0.38 vs 3.84 ± 0.58, P<0.05) in Drp-1 KO mice compared to control. Finally, we observed that the survival rate of Drp-1 KO mice was significantly reduced compared to control mice. Our results demonstrate that inhibition of mitochondrial fission via disruption of Drp-1 inhibits autophagy and causes mitochondrial dysfunction, thereby promoting cardiomyopathy.
Abstract 272: Pharmacological Testing Of Novel Atrial-specific Ion Channel Blockers Establishes Human Pluripotent Stem Cell Derived Atrial Cardiomyocytes As A Predictive Pre-clinical Model For Selective Pharmacology
Authors: Devalla Harsha Deepti , Verkerk Arie O , Passier Robert
Published: July 2021

Background:
Atrial Fibrillation (AF) is the most prevalent arrhythmia affecting two and half million people in the United States alone. Medical therapy is the choice of treatment in a large number of cases and current drug discovery programs by pharmaceutical industries are aimed at developing atrial-specific ion channel blockers. Most of the current selective pharmacology studies however, are carried out in animal models such as dogs and guinea pigs. Human pluripotent stem cell derived cardiomyocytes provide a promising alternative in this respect.

Aim:
To establish an in vitro pre-clinical model of selective pharmacology for testing atrial-specific pharmacological compounds using human pluripotent stem cell (hPSC) derived atrial-like cardiomyocytes.

Methods & Results:
We have successfully generated atrial-like cardiomyocytes from differentiating human embryonic stem cells by modulating retinoic acid signaling. Molecular identity of these cardiomyocytes was established by whole genome microarray while the functional identity was confirmed by voltage patch clamp techniques. At the electrophysiological level, hPSC-derived atrial-like cardiomyocytes exhibit shorter action potentials, increased Ito1 density and increased steady state currents (combination of IKr, IKs and IKur) as expected for atrial cardiomyocytes. In order to validate the use of these cells for selective pharmacology, novel IKur and IKACh blockers (currently in clinical trials/pre-clinical development) were tested and their effect was assessed by electrophysiology. hPSC-derived atrial-like cardiomyocytes showed prolongation of action potential in response to IKur and IKACh blockers whereas the effect was minimal on hPSC-derived ventricular-like cardiomyocytes. These findings are being compared against multiple ion-channel blockers such as Dronaderone.

Conclusions:
Modulation of retinoic acid signaling drives differentiating human pluripotent stem cells to atrial-like cardiomyocytes which have been validated as a pre-clinical screening model for testing novel atrial-specific pharmacological compounds.
Abstract 273: Levels Of Vcam-1 Are Associated With Flow-mediated Dilation In Healthy African American Adults
Authors: LING CHENYI , Williamson Sheara , Diaz Keith , Kretzschmar Jan , Feairheller Deborah , Sturgeon Kathleen , Veerabhadrappa Praveen , Perkins Amanda , Lee Hojun , Grimm Heather , Kashem Abul , Babbitt Dianne , Brown Michael
Published: July 2021

Background:
Vascular cell adhesion molecule-1 (VCAM-1) mediates the adhesion of monocytes to vascular endothelium, which play a role in the development of endothelial dysfunction and atherosclerosis. Flow-mediation dilation (FMD) is an endothelium-dependent response, which is mediated by nitric oxide and closely related to endothelial function. The purpose of the study was to test whether levels of VCAM-1 were associated the endothelial-dependent (FMD) in normotensive African Americans. Normotensives were studied in order to eliminate the influence of hypertension.

Methods and Results:
Blood was collected in 22 subjects aged 41-62 yrs with normal blood pressure (125.0 ± 9.1 mmHg) after 12 hr fast. Plasma was obtained by centrifugation within 15 min after collection in 4 degree. A commercially available solid phase sandwich enzyme linked immuno-sorbent assay kit was used to detect VCAM-1 levels in plasma. FMD was measured after 5-min cuff occlusion using Doppler ultrasound. The maximum FMD diameters were calculated. In the entire study population, levels of VCAM-1 (556.92 ± 154.20 ng/mL) were negatively correlated with the true peak FMD (FMD: 7.81 ± 2.22 %, r=-0.484, P=0.023).

Conclusion:
Levels of VCAM-1 were associated with endothelial responsiveness in healthy African Americans with normal blood pressure. These results suggest that the level of VCAM-1 may be a predictive of endothelial dysfunction in the unique healthy populations.
Abstract 275: Interleukins 17th and 22th in Stable Coronary Artery Disease
Authors: Oliveira Dinaldo , Pitta Maira R , Pitta Ivan R , Heide Elayne , Gomes Viviane R , Filho Edgard V , Victor Edgar G , Oliveira Filho João B
Published: July 2021

Introduction:
The role of the immune and inflammatory pathways in coronary artery disease (CAD) is important but not complete understood. The aim of this study was to evaluate expressions of the interleukins 17th and 22th in patients with stable coronary artery disease.

Hypothesis:
Interleukins 17th and 22th are not increased in stable CAD.

Methods:
This is a cross-sectional, prospective, analytical study, conducted from August to December 2012. We included 40 patients (P) with stable CAD, CCS III or IV, ischemic myocardial scintigraphy, who had not been subjected to any kind of myocardial revascularization and with coronary stenosis equal or major than 50% according to current coronary angiography. There were 20 healthy volunteers (C), to take up comparison of expression of interleukins (IL). We evaluated the levels of IL 17th and 22th of the patients and controls. Interleukins were evaluated in serum of patients and after 48 hours of cells in culture with and without stimulus. IL concentrations were expressed in pg / ml. Statistical analysis was performed using the Mann-Whitney or Student t test. P ≤ 0,05 was considered statistically significant.

Results:
There were 26 men and 14 women in the group of the patients and 12 men and 8 women in the controls. The age was similar between the groups (63.2 ± 8.9 years vs 57.9 ± 9.4, p = ns). The comparison between the groups showed: Interleukin 17th: Serum: P = 3.9 (972.2 -- 2.93) vs C = 3.90 (28.8 -- 1.74), p = 0.5; culture 48 hours without stimulus: P = 3.90 (3.90 -- 3.90) vs C = 6.37 (3.90 - 11), p = 0.8; culture 48 hours with stimulus: P = 302.42 (2200 -- 3.90) vs C = 815 (1353 -- 3.90), p = 0.06. Interleukin 22th: Serum: P = 15.62 (64.72 -- 15.62) vs C = 15.62 (121 -- 15.62), p = 0.2; Culture 48 hours without stimulus: P = 11 (128.93 -- 7.81) vs C = 7.81 (7.81 -- 7.81), P = 0.8; Culture 48 hours with stimulus: P = 135 (2486.7 -- 7, 81) vs C = 322.86 (1319.11 -- 7.81), p = 0.4.

Conclusions:
There were no differences in concentrations of interleukins, but the trend of higher expression of the IL 17th in the controls after cell culture with stimulus. In conclusion, in patients with stable CAD the interleukins 17th and 22th did not exhibit increased concentrations.
Abstract 276: Shockwave Reduces Macrophage Foam Cell Formation Via Increased Expression of ATP Binding Cassette Transporters.
Authors: Cho Wonkyoung , Yoon Young Eun , Kwon Kihwan , Park Young Mi
Published: July 2021

Background:
Excessive lipid accumulation by macrophages plays a crucial role in atherosclerosis. Foam cells are generated by uncontrolled uptake of modified LDL, especially oxidized LDL (oxLDL), and/or impaired cholesterol efflux mediated by ATP-binding cassette (ABC) family transporters, ABCA-1 and ABCG-1. Shockwave, elicited by transient pressure disturbance, have been used for extracorporeal lithotripsy or for treating musculoskeletal disorders. Our current study suggests an evidence that shockwave may have anti-atherogenic effect by inhibiting foam cell formation.

Methods/Results:
Murine peritoneal macrophages were exposed to shockwaves at 0.04 mJ/mm
2
with 1000 impulses, lysed after 6, 18 and 24 hours, and tested for expression of ABCA-1 and ABCG-1. The western blot showed that shockwave induced 2.0-2.8 fold increase of ABCA-1 and ABCG-1 within 18-24 hours. mRNA levels of ABCA-1 and ABCG-1 were also increased by shockwave with 2.0 fold of peak increase in 18 hours. The increased expression of ABCA-1 and ABCG-1 was mediated by phosphorylation of ERK 1/2 (Tyr204). Western blot analysis revealed that shockwave induced phosphorylation of ERK 1/2 (Tyr204) in murine macrophages. Shockwave-induced increase of ABCA-1 and ABCG-1 was blocked by U0126 (40µM), a specific inhibitor for ERK. Oil-red O staining showed that macrophages exposed to shockwave had 25% less intracellular lipid droplets. Intracellular cholesterol measured by cholesterol oxidase and esterase revealed that macrophages exposed to shockwave had 23% less intracellular cholesterol when incubated with oxLDL (50µg/ml) for 16 hours. In vitro migration assays including modified Boyden chamber migration assay and scratch wound healing migration assay showed that macrophages exposed to shockwave had 1.2 fold more migration and had diminished migration-inhibitory effect of oxLDL.

Conclusions:
Shockwave reduces macrophage foam cell formation via ERK-mediated increase of ABCA-1 and ABCG-1 mediating lipid efflux and promotes macrophage migration which may induce macrophage egress from atherosclerotic lesion. Our study suggests anti-atherogenic effects of shockwave as a potential treatment modality for atherosclerosis.
Abstract 279: Loss Of Rad GTPase Produces A Sympathomimetic Cardiac Phenotype Leading To Calcium Overload And Arrhythmia.
Authors: Manning Janet R , Yin Guo , Penn John , Kaminski Catherine , Sievert Gail , Satin Jonathan , Andres Douglas A
Published: July 2021

Background:
Rad is a small GTPase that regulates inward calcium current in excitable cells. In human heart failure, calcium dysregulation is accompanied by lowered levels of Rad expression, and dampened responsiveness to beta-adrenergic stimulation. However, the effects of Rad ablation on calcium cycling in the heart have yet to be examined.

Hypothesis:
Loss of Rad emulates beta-adrenergic stimulation in cardiac myocytes, elevating calcium levels, and promoting after depolarizations.

Methods:
The role of Rad was evaluated using knockout mice (RadKO)at several levels of biological organization. Inward calcium current, action potentials, fractional shortening, and calcium transients were measured in isolated ventricular myocytes. Functional parameters were recorded in the isolated working heart, and telemetry was used to monitor ECGs in intact freely-roaming mice.

Results:
Isolated ventricular myoctyes from Rad null mice display increased ICaL density, greater inward current at low voltages, and after depolarizations at low frequencies. Isolated cells exhibit increased diastolic calcium levels and increased dynamic calcium changes in transient amplitude during pacing. Further, RadKO cardiomyocytes develop a significantly greater number of spontaneous calcium transients than wildtype cohorts. Ventricular myocytes and intact working hearts from RadKO mice fail to respond to beta-adrenergic stimulation at the level of channel activation, calcium transient kinetics, and exhibit a significantly dampened change in +dP/dt in the isolated heart. Consistent with these effects, PKA substrates are phosphorylated at baseline in RadKO cardiomyocytes, suggesting tonic PKA activation.

Conclusion:
These data suggest the new provocative hypothesis that beta-adrenergic-mediated changes in excitation, calcium handling, and heart contraction involve relief of Rad-dependent negative regulation of channel function.
Abstract 281: Studying Structural And Contractility Defects Of Cardiomyocytes In An In Vitro Model Of Human Hypertrophic Cardiomyopathy
Authors: Ribeiro Marcelo C , van Hoorn Hedde , Monshouwer-Kloots Jantine J , Mummery Christine L , Schmidt Thomas , Passier Robert
Published: July 2021

Hypertrophic Cardiomyopathy (HCM) is a cardiac disease, morphologically characterized by cardiac hypertrophy, fibrosis and impaired heart function. HCM is primarily associated with mutations in sarcomeric proteins. Mutations in sarcomeric Myosin Binding Protein C (MYBPC3) account for approximately 25% of all HCM patients. The majority of mutations in MYBPC3 are nonsense mutations, predicted to produce truncated proteins. However, instead of truncated proteins, lower levels of full-length protein of MYPBC3 are frequently found in heart samples of patients with mutations in MYBPC3, strongly suggesting that haploinsufficiency is responsible for the cardiac disease phenotype. It has been previously shown that upon ablation of MYBPC3 the myosin cross-bridges are localized closer to the actin filament, suggesting that MYBPC3 functions as a structural restraint, holding a fraction of the myosin heads at a specific distance from the actin filament. We hypothesize that the shortening of the distance between the myosin cross-bridges and the actin filaments is responsible for the increase of stretch activation kinetics leading to an increase in calcium sensitivity and contractile dysfunction. In order to develop a human in vitro model for HCM we mimicked MYBPC3 haploinsufficiency by generating stable lines in human pluripotent stem cells-derived cardiomyocytes (hPSC-CM) in which MYPBC3 protein levels were reduced by 40 to 90% using short-hairpin RNA sequences. To analyze structural and Ca2+ kinetic changes induced by the MYBPC3 knockdown we are using stochastic optical reconstruction microscopy (STORM), a superresolution fluorescence imaging method, to measure the distances between sarcomeric proteins in combination with Ca2+ imaging using a ratiometric method. At a mechanical level we are able to measure total cell force of contraction by measuring the deflection of pillars on a micropillar array generated by control and MYBPC3 knockdown hPS-CM during contraction. In conclusion, we successfully developed a human in vitro model for cardiac MYBPC3 haploinsufficiency in combination with state of the art biophysical and imaging techniques, which will allow us to comprehend the molecular mechanisms responsible for the development of HCM.
Abstract 286: Structural Determinants Of Thermodynamic Stability And Actin-binding Function Of Dystrophin And Utrophin
Authors: Mallela Krishna , Bandi Swati , Singh Surinder
Published: July 2021

Muscular dystrophy (MD) is an incurable disease, and affects all types of muscles. The decreased function of heart muscles causes heart problems such as cardiomyopathy and congestive heart failure. A deficiency in functional dystrophin protein in muscle cells triggers the onset of Duchenne MD and Becker MD. Utrophin is the closest homologue of dystrophin and is being tested as a protein drug to replace the loss of functional dystrophin in human patients. However, utrophin is less stable and binds to actin through a different mode of contact when compared with dystrophin. To optimize utrophin as the protein drug, we first need to understand how its molecular structure controls its stability and function. Utrophin and dystrophin bind to actin using tandem calponin-homology (CH) domains at their N-terminus. Individual CH domains of utrophin and dystrophin are very similar in amino acid sequence and their three dimensional structures. The major difference is in their relative orientation around the linker region that connects the two CH domains. To determine the role of the linker, we designed constructs with linkers switched between utrophin and dystrophin. Our results indicate that the tandem CH domain of utrophin with dystrophin linker (UDL) is more stable than that of utrophin but less stable than that of dystrophin. Similarly, tandem CH domain of dystrophin with utrophin linker (DUL) is less stable than that of dystrophin but more stable than that of utrophin. Kinetic folding and unfolding studies suggest that the linker predominantly affects the folding rate rather than the unfolding rate. In addition to stability and folding, the linker also controls protein aggregation. UDL is more prone to aggregation when compared with that of utrophin, whereas DUL is less prone to aggregation when compared with that of dystrophin. These results indicate that the linker plays a major role in controlling the stability and function of tandem CH domains, and further suggests that it is possible to engineer utrophin to behave precisely like dystrophin based on their molecular structures.
Abstract 288: Mice With Disruptive Egfr Signaling In The Aortic Valves Develop Calcific Aortic Valve Stenosis
Authors: Zhou Bin , Wu Bingruo
Published: July 2021

Calcific aortic valve stenosis is a major health problem. Despite its clinical importance, the pathogenesis of this condition remains illusive. Aortic valves are made of endothelial cells and interstitial cells, both are derived from the endocardial cells of the heart during development. We have developed a new mouse model, Nfatc1-Cre, to specifically study the biology of the valve cells during valve development. In this study, we deleted epidermal growth factor receptor (Egfr) in the aortic valves using the Nfatc1-Cre and showed by histological and biochemical analyses that this deletion caused a spectrum of pathological characteristics of human calcific aortic valve stenosis. They include increased proliferation and dedifferentiation of the valve interstitial cells, abnormal deposition of extracellular matrix, bone-like formation and calcification. The null mice eventually developed the left ventricular dysfunction, presumably secondary to the stenotic aortic valves. We also revealed by immunohistochemistry and quantitative RT-PCR the changes in gene expression involved in the epithelial-to-mesenchymal transition or osteogenic activities. Altogether, these results demonstrate that Egfr signaling in the valve endothelial and interstitial cells regulates the homeostasis of the aortic valves and suggest that patients with mutations or medications affecting the Egfr signaling may be at the risk to develop calcific aortic valve stenosis. Our study also provides the proof-of-principle data showing that the Nfatc1-Cre is a useful genetic tool to elucidate the valve-specific gene function involved in the valve homeostasis and disease.
Abstract 290: Haemogenic Endocardium Contribute To Definitive Hematopoiesis During Cardiogenesis
Authors: Nakano Haruko , Liu Xiaoqian , Arshi Armin , van Handel Ben , Sasidharan Rajkumar , Harmon Andrew W , Shin Jae-Ho , Schwartz Robert J , Conway Simon J , Harvey Richard P , Pashmforoush Mohammad , Mikkola Hanna K , Nakano Atsushi
Published: July 2021

The circulatory system is the first functional organ system that develops during mammalian life. Accumulating evidences suggest that cardiac and endocardial cells can arise from a single common progenitor cell during mammalian cardiogenesis. Notably, these early cardiac progenitors express multiple hematopoietic transcription factors, consistent with previous reports. Indeed, a close relationship among cardiac, endocardial and hematopoietic lineages has been suggested in fly, zebrafish, and embryonic stem cell in vitro differentiation models. However, it is unclear when, where and how this hematopoietic gene program is in operation during in vivo mammalian cardiogenesis. Hematopoietic colony assay suggests that mouse heart explants generate myeloids and erythroids in the absence of circulation, suggesting that the heart tube is a de novo site for the definitive hematopoiesis. Lineage tracing revealed that putative cardiac-derived Nkx2-5+/Isl1+ endocardial cells give rise to CD41+ hematopoietic progenitors that contribute to definitive hematopoiesis in vivo and ex vivo during embryogenesis earlier than in the AGM region. Furthermore, Nkx2-5 and Isl1 are both required for the hemogenic activity of the endocardium. Together, identification of Nkx2-5/Isl1-dependent hemogenic endocardial cells (1) adds hematopoietic component in the cardiogenesis lineage tree, (2) changes the long-held dogma that AGM is the only major source of definitive hematopoiesis in the embryo proper, and (3) represents phylogenetically conserved fundamental mechanism of cardio-vasculo-hematopoietic differentiation pathway during the development of circulatory system.