bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 23: Overexpression Of Tbx20 In Adult Cardiomyocytes Promotes Cardiomyocyte Proliferation And Improves Cardiac Function Post Myocardial Infarction
Authors: Xiang Fu-li , Yutzey Katherine
Published: July 2021

Background:
Adult mammalian cardiomyocytes (CM) have the potential to proliferate, but this is not sufficient to compensate for the massive loss of functional CMs after myocardial infarction (MI), which remains a leading cause of death in the US. During embryonic heart development, the transcription factor Tbx20 is required for CM proliferation, and Tbx20 overexpression promotes fetal characteristics in adult CMs when initiated before birth in mice. We hypothesize that Tbx20 overexpression (Tbx20OE), when induced in adult CMs after injury, improves cardiac function and repair via dedifferentiation of CMs, thus promoting cell cycle re-entry and repair in mice post-MI.

Methods and Results:
αMHCMerCreMer (STG) and the inducible cardiomyocyte-specific Tbx20 transgenic (αMHCMerCreMer/CAG-CAT-Tbx20, DTG) mice were subjected to MI or sham surgeries. Tbx20OE was induced 3 days post-surgery via tamoxifen to specifically target cardiac repair post-MI. In sham-operated mice, no difference in cardiac function or morphology was observed between DTG and STG groups. However, more proliferating CMs as labeled by Ki67 were found in DTG sham myocardium compared to STG. Expression of cyclin D1, E1 (cell cycle markers) and IGF1 mRNA was increased, while p21 (cell cycle inhibitor) and Meis1 (negative regulator of proliferation) were decreased, in DTG sham hearts compared to STG controls. In mice subjected to MI, cardiac function, as measured by echocardiography, was significantly improved, and the infarct scar size was smaller (58.1% vs 38.3%) in the DTG group compared to STG controls 2 and 4 weeks post-MI. Myocardial hypertrophy determined by heart to body weight ratio and myocyte diameter was also significantly reduced in DTG heart compared to STG 4 weeks post-MI. Thus, induction of Tbx20OE post-MI injury leads to improved cardiac performance, decreased scar size, and decreased maladaptive cardiac remodeling. Ongoing studies will determine if proliferation indices (Ki67, pHH3, aurora kinase B) and cytokinesis of CM post-MI are increased in myocardium and isolated adult cardiomyocytes with Tbx20OE.

Conclusions:
Tbx20OE in adult CM activates cell proliferation markers and also improves cardiac function and repair in mice when induced post-MI.
Abstract 237: Intronic 27-nt Mirna Suppresses The Expression Of Endothelial Nitric Oxide Synthase Predominantly Through Nuclear Transcription Factor Ap-1
Authors: Liu Patrick , Yan Limei , Li Meimei , Hao Hong , Liu Zhenguo , Ou Hesheng
Published: July 2021

The 27-base pair (bp) repeats in intron 4 were considered as the source of small RNA that suppressed endothelial nitric oxide synthase (eNOS) expression in endothelial cells. The present study was to investigate the role of polymorphic intronic 27-nucleotide (nt) miRNA and nuclear transcription factors (TFs) in eNOS expression in endothelial cells. The 27-nt or 54-nt miRNA or their mutants (with 3 mutations at 13, 14, and 15
th
nt for 27-nt mutant and at 13,14,15,40,41,42
th
nt for 54-nt mutant) were stably transfected and expressed in human aortic endothelial cells (HAECs). Overexpression of 27-nt miRNA and its duplexes and mutants significantly decreased eNOS mRNA level and protein content. Interestingly, more reduction in eNOS protein was observed in the cells overexpressing the mutant miRNAs than the cells overexpressing the wild-type ones for both 27-nt and 54-nt miRNAs. Western blotting showed that transcription factor Ap-1 expression was substantially decreased in the cells that overexpressed either 27-nt or 54-nt miRNAs (both wild-type and mutants). Augmented inhibition of Ap-1 expression was observed in the cells overexpressing the mutant 27-nt miRNA as compared with the wild-type. However, Sp-1 protein expression was significantly suppressed only in the cells overexpressing 54-nt miRNA. Silencing Ap-1 abolished the polymorphic effect of miRNAs both in length and mutation on eNOS expression; while Sp-1 silencing only reversed the polymorphic effect of mutant miRNAs. These results suggested that overexpression of intron 4-based 27-nt miRNAs suppressed eNOS gene expression polymorphically through interactions predominantly with nuclear transcription factors Ap-1 in HAECs.
Abstract 239: Selective Class I and II Histone Deacetylation Inhibitors Alter Stability of HDAC-Corepressor Complexes
Authors: Wang Hsiao C , Harris Lillianne G , Chou James C , Mani Santhosh , Menick Donald
Published: July 2021

Introduction:
Alterations in expression and activity of different genes have been implicated in the pathogenesis of heart failure. Our lab has shown that HDAC-repressor complexes play a critical role in the upregulation Sodium Calcium Exchanger (
Ncx1)
and HDAC inhibition causes changes that attenuated cardiac remodeling during cardiac hypertrophy and heart failure. Thus, treatment with HDAC inhibitors has been proposed as a potential strategy for treatment of cardiac hypertrophy and heart failure. HDAC inhibitors repress deacetylase activity but we propose that they also affect HDAC confirmation and interaction with other protein factors. We hypothesize that HDAC inhibitors affect the stability of the co-repressor complex with specific transcription factors and that this effect is dependent on the transcription factor.

Results:
Inhibition of HDACs in adult cardiomyocytes results in the greater stabilization of HDACs with co-repressor molecules that were recruited to the NCX1 promoter through Nkx2.5 transcription factor. HDAC class I specific inhibitor, MS 275 demonstrated stronger association between HDACs and co-repressors while other Class I inhibitors, PD106 and BML 210 failed on showing this phenomenal. The results suggested that class I HDACs inhibitors may affect formations of HDAC-complex via alternated active site interactions other than chelating with zinc binding domain. These results compliment ChIP experiments which also demonstrate the different recruitments of Sin3a at the proximal promoter of NCX1.
In vivo
analysis on HDAC5 knockout mice reveal that the Sin3a-HDAC1/2 repressor complex is not recruited to the
Ncx1
promoter in the absence of HDAC5, indicating not only Class I HDAC but also Class II HDACs play an important role on HDAC-complex formation.

Conclusions:
This work gives insight into part of the molecular mechanism of how HDAC inhibitors can affect the stability of the HDAC co-repressor complex in cardiac hypertrophy and heart failure. In addition, we demonstrated the Class IIa HDACs are required for the recruitment of the Sin3a/HDAC1/2 co-repressor complex to specific transcription factors on the target promoter.
Abstract 240: Vascular Differentiation of C-kit+ Cardiac Progenitor Cells in Bioactive PEG Hydrogels
Authors: Bhutani Srishti , French Kristin M , Garcia Andres J , Davis Michael E
Published: July 2021

Ischemic heart disease is the leading cause of death in the United States. The ideal therapy would include regeneration of functional myocardial cells as well as vascularization of the regenerated cardiac tissue. In this context, c-kit+ cardiac progenitor cells (CPCs) isolated from the heart are an exciting stem cell population as they have been shown to have potential to differentiate into cells of myocardial as well as vascular (endothelial, vascular smooth muscle) lineages. Being an autologous adult stem cell source, they provide advantages of alleviation of immune-rejection and disease transmission risks and are free from ethical concerns. Driving the vascular differentiation of CPCs can enable them to be used for angiogenic cell therapy. The objective of this project is to direct CPCs to the endothelial lineage via stimulation with VEGF immobilized in a PEG-maleimide (PEG-MAL) hydrogel scaffold for better retention of grafted cells. This hydrogel has protease-cleavable sites which should enable the hydrogel to be degraded while allowing for tube formation. CPCs are encapsulated in PEG-MAL hydrogel constructs presenting 100 ng VEGF/mL hydrogel. CPCs encapsulated in these PEG-MAL hydrogels maintain high viability for up to 14 days. In hydrogels presenting immobilized VEGF, successful biochemical stimulation of the encapsulated CPCs is evidenced by downstream ERK phosphorylation, likely through VEGFR2. These immobilized-VEGF treated CPCs also demonstrate greater RNA expression of endothelial markers 7 days post-encapsulation. CPCs in VEGF presenting gels show a trend toward increasing formation of vascular structures in comparison to cells encapsulated in empty hydrogels. Together, this preliminary evidence suggests that c-kit+ cardiac progenitor cells can be driven toward the endothelial lineage when stimulated with immobilized VEGF, and may adapt a vascular phenotype. This system has the potential to be used for therapeutic angiogenesis as the PEG hydrogel scaffold should enable controlled delivery of the CPCs in vivo and is injectable and biodegradable.
Abstract 256: Paracrine miRNA-Crosstalk Between Human CD34+ Stem Cells and Selective Myocardial Cells Via Therapeutic Exosomes Promotes Repair of the Ischemic Heart
Authors: Sahoo Susmita , Kim David , Misener Sol , Kamide Christine E , Vaughan Douglas E , Losordo Douglas W
Published: July 2021

Introduction:
Earlier, in a first study of its kind, we have demonstrated a novel mechanism that therapeutically significant human CD34+ stem cells secrete membrane bound nano-vesicles called exosomes (CD34Exo). CD34Exo are angiogenic and constitute a critical component of the pro-angiogenic paracrine activity of the cells. Further, when transplanted locally, cell-free CD34Exo induce ischemic tissue repair in a murine hindlimb ischemia model.

Here, we hypothesize that exosomes released via paracrine secretion from human CD34+ cells mediate myocardial repair by direct transfer of microRNAs to target cells in the heart.

Methods and Results:
When injected into mouse ischemic myocardium, cell-free CD34Exo replicated the therapeutic activity of human CD34+ cells by significantly improving ischemia (ejection fraction, 42±4 v 22±6%; capillary density, 113±7 v 66±6/HPF; fibrosis, 27±2 v 48±7%; p<0.05, n=7-12) compared with PBS control. Interestingly, confocal imaging and flow cytometry analyses of the exosomes-injected ischemic myocardial tissue revealed that CD34Exo was selectively internalized into endothelial cells and cardiomyocytes. CD34Exo, which is enriched with miR126, induced the expression of miR126 and several pro-angiogenic mRNAs in the exosomes-treated ischemic myocardium, but did not affect the endogenous synthesis of miR126. CD34Exo lacking miR126 had decreased angiogenic activity in vitro and decreased proangiogenic gene expression in vivo indicating that miR126 is important for CD34Exo function. Imaging using fluorescent miR126 confirms that CD34Exo directly transferred miR126 and possibly other yet to be identified moieties from its cargo, selectively to endothelial cells and cardiomyocytes in the ischemic heart.

Conclusion:
Our results reveal a novel molecular and trafficking mechanism of CD34Exo that may be responsible for intercellular transfer of genetic information such as miRNAs from human CD34+ stem cells, selectively to endothelial cells and cardiomyocytes inducing changes in gene expression, angiogenesis and myocardial recovery. Exosomes-shuttled miRNAs may signify amplification of stem cell function and may explain the therapeutic benefits associated with human CD34+ cell therapy.
Abstract 257: Clonal Analysis Of Multipotent Cardiovascular Progenitors That Generate Cardiomyocytes During Development
Authors: Sereti Konstantina Ioanna , Kamran Rashani Paniz , Zhao Peng , Ardehali Reza
Published: July 2021

It has been proposed that cardiac development in lower vertebrates is driven by the proliferation of cardiomyocytes. Similarly, cycling myocytes have been suggested to direct cardiac regeneration in neonatal mice after injury. Although, the role of cardiomyocyte proliferation in cardiac tissue generation during development has been well documented, the extent of this contribution as well as the role of other cell types, such as progenitor cells, still remains controversial. Here we used a novel stochastic four-color Cre-dependent reporter system (Rainbow) that allows labeling at a single cell level and retrospective analysis of the progeny. Cardiac progenitors expressing Mesp1 or Nkx2.5 were shown to be a source of cardiomyocytes during embryonic development while the onset of αMHC expression marked the developmental stage where the capacity of cardiac cells to proliferate diminishes significantly. Through direct clonal analysis we provide strong evidence supporting that cardiac progenitors, as opposed to mature cardiomyocytes, are the main source of cardiomyocytes during cardiac development. Moreover, we have identified quadri-, tri-, bi, and uni-potent progenitors that at a single cell level can generate cardiomyocytes, fibroblasts, endothelial and smooth muscle cells. Although existing cardiomyocytes undergo limited proliferation, our data indicates that it is mainly the progenitors that contribute to heart development. Furthermore, we show that the limited proliferation capacity of cardiomyocytes observed during normal development was enhanced following neonatal cardiac injury allowing almost complete regeneration of the scared tissue. However, this ability was largely absent in adult injured hearts. Detailed characterization of dividing cardiomyocytes and proliferating progenitors would greatly benefit the development of novel therapeutic options for cardiovascular diseases.
Abstract 276: The Regulatory Effects Of Peroxiredoxin II On Phospholamban Phosphorylation And Cardiac Contractile Function In Vivo
Authors: Zhao Wen , Shi Xiaojing , Zhou Wenjuan , Wang Huimin , Geng Xuepeng , Zhang Shuang , Wang Jingjing , Kranias Evangelia G , Liu Hong-Min
Published: July 2021

Peroxiredoxin II (prxII), a cytosolic form of the anti-oxidant peroxiredoxin family, was recently found to be decreased in failing human hearts. Interestingly, in hyperdynamic hearts of two genetically modified mouse models with: a) phospholamban ablation; and b) overexpression of the active inhibitor-1 of protein phosphatase 1, the levels of this cellular peroxidase (prxII) were markedly increased. Acute overexpression of prxII by adenoviral-delivery in adult rat cardiomyocytes (Ad-prxII) was associated with decreases in the basal rates of contraction and relaxation, as well as calcium kinetics. Accordingly, Ad-prxII-AS infected cardiomyocytes exhibited enhanced contractile parameters and Ca-kinetics. The depressed or increased contractility by Ad-prxII or Ad-prxII-AS was associated with parallel decreases or increases in phosphorylation of phospholamban (Ser16 and Thr17). To determine the in vivo effects of prxII on cardiac contractility, three transgenic lines (TG) with 2-3 fold cardiac-specific overexpression of prxII were generated and their cardiac morphologic and functional phenotypes were characterized. The TG mice exhibited no alterations in cardiac pathology or morphology up to 4 months of age. However, langendorf perfusions revealed that cardiac contractility, including the rates of contraction and relaxation (±dp/dtmax) as well as the left ventricular end systolic pressure (LVESP), were significantly depressed in TG mice (to 75, 76 and 63%, respectively), compared to WTs (100%). The depressed function was not associated with any alterations in the expression levels of key SR calcium handling proteins: SERCA2, total phospholamban, calsequestrin and ryanodine receptor. However, the levels of the phosphorylated PLN at Ser16 were found to be reduced to 50% in the TG mice, compared to WTs. These findings indicate that prxII, an anti-oxidant protein, may regulate basal cardiac contractile performance in vivo through phospholamban phosphorylation.
Abstract 286: Myocardin-related Transcription Factors Control Epicardial EMT and Coronary Vessel Homeostasis
Authors: Trembley Michael A , Velasquez Lissette S , Hoffman Corey M , de Mesy Bentley Karen L , Small Eric M
Published: July 2021

The epicardium is a source of multi-potent progenitor cells that envelop the heart and contribute to various cardiac lineages through the process of epithelial-to-mesenchymal transition (EMT). Although known upstream cues promote epicardial EMT via changes in gene expression and actin cytoskeletal dynamics, the link between these signals and progenitor cell motility remain unclear. Myocardin-related transcription factor (MRTF) cofactors are primarily retained in the cytoplasm through interactions with G-actin. However, upon cytoskeletal reorganization and G-actin depletion, nuclear MRTFs associate with serum response factor (SRF) to drive cellular motility and contractility gene programs reminiscent of EMT. Here, we identify the SRF/MRTF gene regulatory axis as a key mediator of epicardial EMT. We found significant enrichment of MRTF-A and -B in the epicardium prior to EMT. MRTFs were later expressed in a spatial and temporal manner concurrent with EMT and epicardial-derived cell (EPDC) differentiation. Furthermore, MRTF deletion attenuates contractile gene expression in epicardial explants and impairs migration of EPDCs into subjacent cell layers using
ex vivo
assays. Epicardial EMT and EPDCs differentiation are necessary for proper coronary vessel formation. We found that genetic ablation of MRTFs results in disrupted coronary plexus formation, endothelial cell dysfunction, and sub-epicardial hemorrhage. The vascular phenotype observed in MRTF-A/B
epiDKO
mice results in part from the depletion (~50% reduction) of epicardial-derived coronary pericytes. These data suggest a critical role for MRTFs in coronary vessel formation by regulating epicardial EMT and mobilizing EPDCs.
Abstract 288: Rnd3/RhoE Regulates Cardiac Ryanodine Receptor Type 2 Stability
Authors: yang xiangsheng , Wang Tiannan , Yue Xiaojing , Wehrens Xander H.T. , Chang Jiang
Published: July 2021

Rationale:
Rnd3, a small Rho GTPase, is involved in the regulation of cell actin cytoskeleton dynamics, cell migration and proliferation. The biological function of Rnd3 in the heart remains unexplored.

Objective:
To define the functional role of the
Rnd3
gene in the animal heart and investigate the associated molecular mechanism.

Methods and Results:
By loss-of-function approaches, we discovered a new role in which Rnd3 stabilizes the ryanodine receptor type 2 (RyR2) Ca
2+
release channel. Genetic deletion of Rnd3 in mice resulted in embryonic lethality with heart failure and arrhythmia. Both Rnd3
-/-
embryonic and Rnd3
+/-
adult cardiomyocytes showed severe Ca
2+
leakage. Single channel assessment showed the destabilized RyR2 channel, and this irregular spontaneous Ca
2+
release was curtailed by protein kinase A (PKA) inhibitor treatment. Further studies found that RyR2 protein was hyperphosphorylated by PKA in the mutant heart. Remarkable increases in the PKA activity along with elevated cyclic adenosine monophosphate levels were detected
in vivo
in Rnd3-null embryos and
in vitro
in neonatal rat cardiomyocytes and non-cardiac cell lines with Rnd3 knockdown. Moreover, we found increasing β
2
-adrenergic receptor (β
2
AR) protein levels, but no correlated mRNA changes in both the Rnd3-null heart and non-cardiac cells with Rnd3 knockdown. Immunoprecipitation analysis demonstrated that Rnd3 and β
2
AR physically interacted. Multiple post-translational modification analyses of β
2
AR revealed that downregulation of Rnd3 attenuated β
2
AR protein lysosomal targeting and ubiquitination, which in turn resulted in the elevation of β
2
AR protein levels contributing to the activation of PKA signaling. Rnd3 deficiency had no effects on the hydroxylation- and sumoylation-mediated β
2
AR protein degradation.

Conclusion:
Rnd3 is a unique stabilizer of RyR2 that impacts intracellular Ca
2+
handling in the heart.
Abstract 29: Novel Atheroma-specific Atheroprotective Factor Promotes VSMC Pro-survival Signaling
Authors: Segedy Amanda K , Pyle Amy L , Li Bin , Zhang Youmin , Babaev Vladimir , Jat Parmjit , Fazio Sergio , Atkinson James B , Linton MacRae F , Young Pampee P
Published: July 2021

Cardiovascular disease, the leading cause of death in the United States, is primarily driven by atherosclerosis. In recent years, studies have focused on identification of naturally expressed, atheroprotective genes for use in delaying development of or preventing complications from atherosclerosis. To this end, we previously identified SPRR3 (small proline rich protein 3) as a gene specifically upregulated in vascular smooth muscle cells (VSMCs) of atheroma versus healthy arterial tissue of humans and mice. In the present study, we generated ApoE-null mice lacking SPRR3, which displayed significantly increased atheroma burden compared with ApoE-null controls. To determine the cellular driver(s) of this phenotype, we investigated SPRR3-dependent changes in bone marrow-derived cells, endothelial cells (ECs), and VSMCs. Bone marrow transplant of SPRR3-expressing cells into SPRR3-/-ApoE-/- recipients failed to rescue atheroma burden. Similarly, no change was observed in SPRR3-deficient versus control ECs. However, apoptosis was significantly reduced in SPRR3-overexpressing VSMCs in vitro. A positive association was also observed between SPRR3 expression and PI3K/Akt activity in VSMCs. The SPRR3-dependent survival advantage observed in SPRR3-overexpressing cells was lost following treatment with PI3K/Akt pathway inhibitor. Our data indicate that SPRR3 protects the atheroma from VSMC loss by promoting survival signaling in lesion VSMCs, thereby modulating atherosclerosis development. As the first identified atheroma-specific VSMC pro-survival factor, SPRR3 represents a potential target for lesion-specific modulation of VSMC survival to inhibit plaque progression.
Abstract 297: Paroxysmal And Sustained Atrial Fibrillation In A New Large Animal Model Of Nonischemic Heart Failure
Authors: Sikanderkhel Saad , Onibile Olawale , Walcott Gregory P , Pogwizd Steven M
Published: July 2021

Introduction:
Atrial fibrillation is common in heart failure (HF). Understanding of the mechanisms of atrial fibrillation (AF) is limited by the paucity of large animal AF models, especially in the failing heart. We developed a large animal model of nonischemic heart failure (HF) in dogs by combined aortic insufficiency and aortic constriction and observed that a number of HF dogs developed paroxysmal AF on holter monitor. Here we characterize the spontaneously-occurring pAF in these HF dogs and perform electrophysiologic (EP) assessment of atrial refractoriness and AF inducibility along with echocardiographic imaging of left ventricle (LV) and left atrium (LA).

Methods:
HF was induced in dogs by aortic insufficiency and aortic constriction, and serial echocardiography (for LV fractional shortening (FS) and LA size) and Holter monitoring was performed. In control and HF dogs, EP study of atrial refractory period (AERP) and AF inducibility (duration and atrial cycle length (CL)) was performed.

Results:
By Holter monitoring, paroxysmal AF was noted in 5 dogs with episodes ranging from 15 to 94 beats long (mean of 49±27 beats, n=12). In EP studies, control dogs (N=3) exhibited AERP of 176±8 ms. Burst pacing resulted in AF of very brief duration (mean 32±24 sec) and a mean AF CL of 138±6 ms. LV FS averaged 37% and LA size averaged 4.3 cm2. HF dogs (N=5) exhibited RAERP of 150±8 (p=0.05 vs control). Two of these dogs had sustained AF with ventricular response up to 230 bpm on Holter monitor. In the other 3 HF dogs, burst pacing induced AF with a mean duration of 232±185 sec (at times with conversion to atrial flutter) and with a mean AF CL = 110±4 ms (p=0.002 vs control). Echo data showed LVFS averaged 30% and LA area of 14.9 cm2 (p=0.05 vs control).

Conclusion:
Thus we have developed a novel large animal model of HF that exhibits paroxysmal and sustained AF. This model will provide an opportunity for the study of underlying AF mechanisms, the progression of remodeling in HF hearts leading to AF, and the assessment of human-scale interventions to better treat and prevent this arrhythmia.
Abstract 300: The Actin Cytoskeleton Confers Specificity of Cx43 Delivery to Intercalated Discs
Authors: Zhang Shan-Shan , Hong SoonGweon , Lee Luke P , Shaw Robin M
Published: July 2021

Connexin 43 (Cx43) gap junctions (GJs) electrically couple ventricular cardiomyocytes at the intercalated disc (ID), orchestrating organized organ level contraction with each heartbeat. Disease-related disruption of the Cx43 cytoskeletal trafficking machinery is associated with mislocalization of the Cx43 gap junction protein away from the ID and lethal arrhythmias. We recently found that the majority of intracellular Cx43 cargo is associated with actin, not microtubules, and is either paused or moving slowly. It is not understood why actin is involved in Cx43 trafficking. Using micropatterned HeLa cell pairs and whole-cell automated single particle tracking algorithms, we detected that distinct actin polarity exists in the cell, including highly oriented long fibers associated with fast-moving Cx43 cargo aligned toward actively forming GJ plaques. F-actin disruption with latrunculin A (LatA) leads to a loss of Cx43 cargo directionality toward the cell-cell border, as well as a marked decrease in overall microtubule length. We also found a LatA-dependent biochemical interaction between β-actin and the microtubule plus-end-binding protein EB1, which leads growing microtubules and is a necessary component of the Cx43 forward trafficking machinery. In live cell pairs, F-actin disruption resulted in a decrease in overall EB1 activity and in the number of fully extended microtubules that reach the cell-cell border. Together, these results indicate that actin contributes to the specificity of Cx43 delivery by directing EB1-based microtubule growth toward the cell-cell junction (Please refer to attached diagram).
Abstract 317: Beta3 Integrin-mediated CTGF Expression in Cardiac Hypertrophy
Authors: Sundararaj Kamala P , Pleasant Dorea L , Balasubramanian Sundaravadival , Kuppuswamy Dhandapani
Published: July 2021

Connective Tissue Growth Factor (CTGF/CCN2) is a fibrotic mediator overexpressed in human atherosclerotic lesions, myocardial infarction and hypertension. CTGF regulates ECM deposition, fibrosis, wound repair and angiogenesis. Our preliminary studies using wild type (WT) and global 3 integrin knockout (3-/-) mice show for the first time that pressure overload (PO) by transverse aortic constriction (TAC) induces both CTGF and 3 integrin expression in WT mice and that 3 integrin is a prerequisite for the PO-induced CTGF expression. Since both the major cells, cardiomyocyte (CM) and cardiac fibroblast (CFb) have been shown to express CTGF, exploring the primary source for the 3 integrin mediated CTGF expression is expected to identify potential targets for fibrosis. Therefore, the main focus of this proposed study is to use CM specific β3 integrin KO mice with preserved β3-integrin function in CFb as well as CFb specific β3 integrin KO mice with preserved β3-integrin function in CM and evaluate which heart cell type contributes to β3-integrin-mediated PO-induced CTGF production and ECM accumulation. Furthermore, using cells isolated from these mice, we will perform
in vitro
studies to explore the mechanism by which β3-integrin mediates CTGF secretion by analyzing specific nonreceptor tyrosine kinases and transcription factors.
Abstract 334: Proliferation of Cardiomyocytes from a Rat Model of Heart Hypoplasia
Authors: Williams Corin , Wang Raymond , Black Lauren D
Published: July 2021

Decreased cardiomyocyte proliferation has been implicated in underdevelopment of the left ventricle in Hypoplastic Left Heart Syndrome (HLHS). However, it is not clear if cardiomyocytes in HLHS have an inherent defect that inhibits proliferation or if it is the result of abnormal external cues (eg, alterations to mechanical loading or extracellular matrix). We hypothesized that cardiomyocytes from hypoplastic hearts would be capable of proliferation if removed from the unhealthy cardiac environment. We used the rat model of congenital diaphragmatic hernia (CDH) via nitrofen treatment in the pregnant dam at E10 to induce heart hypoplasia in the developing fetuses; control dams were gavaged with olive oil only. Fetuses were harvested at E21 and the presence of CDH was determined. CDH+ hearts were smaller and abnormally shaped compared to healthy controls (
Fig. 1A
). Ki67 staining revealed that CDH+ hearts had significantly lower cell proliferation vs. healthy hearts (
Fig. 1B
). However, when cells were placed in culture, CDH+ cardiomyocytes expanded more rapidly compared to control cardiomyocytes (
Fig. 1C
). CDH+ cells also had smaller, more disorganized sarcomeres (not shown) and decreased expression of cardiac genes compared to controls (
Fig. 1D
), suggesting that CDH+ cells were less mature. Our findings suggest that cardiomyocytes from hypoplastic hearts may be arrested in an immature state but are not necessarily defective, and have potential for proliferation and growth. Strategies that aim to increase cardiomyocyte proliferation during development in HLHS may be a novel strategy to encourage left ventricular growth and lead to better outcomes in these patients.
Abstract 341: GsMTx4-D is a Cardioprotectant against Myocardial Infarction during Ischemia and Reperfusion
Authors: Li Ji , Wang Jinli , Sachs Frederick , Suchyna Thomas M
Published: July 2021

Rationale:
GsMTx4 peptide is a selective inhibitor of cation selective mechanosensitive ion channels (MSCs) and has helped establish the role of MSCs in cardiac physiology. However, the role of MSCs in ischemic reperfusion injury was unknown. Cation imbalance appears to be a major contributor to ischemic reperfusion injury across multiple systems. MSCs may be significant contributors to the cation overload, and they are likely to be activated in reperfusion due to inhomogeneous local stresses and compromised mechanical support of the sarcolemma.

Objective:
The aim of this study was to determine if the D enantiomer of GsMTx4 can act as a cardioprotectant during ischemia/reperfusion in mice.

Methods and Results:
GsMTx4-D pharmacokinetics in the plasma and heart was monitored over 24 hrs using an LCMSMS assay. Ischemia and reperfusion in the mouse heart involved ligating a coronary artery for 20 min followed by release of the ligature. GsMTx4-D was administered by either acute intravenous injection during the ischemic event, or a day prior to the event with two days of intraperitoneal injections, once per day. Dosing of GsMTx4-D was adjusted to achieve tissue concentrations of 1-5 μM. Relative to vehicle injected animals, GsMTx4-D reduced infarct area by >40% for both acute and pretreated animals. Cardiac output was indistinguishable between sham-treated control hearts and GsMTx4-D pretreatment, and arrhythmias were also significantly reduced by intravenous injections of GsMTx4-D.

Conclusions:
GsMTx4-D is a potent cardioprotectant that decreases infarct area, increases cardiac output and decreases arrhythmias that are caused by ischemia and reperfusion. GsMTx4-D was not toxic and promises to be a useful therapy in reperfusion of the heart and other organs.
Abstract 342: Implication Of Mir-98 In The Cardiac Vulnerability Of Pregnancy To Ischemia Reperfusion Injury
Authors: Li Jingyuan , Arany Zoltan Pierre , Eghbali Mansoureh
Published: July 2021

We have recently reported that the late-pregnant(LP) rodent is more prone to myocardial ischemia-reperfusion (I/R) injury compared to non-pregnant(NP). However, the underlying molecular mechanisms involved in the higher susceptibility of LP to IR injury are not quite clear. The objective of this study was to investigate the role of miRNA-98(miR-98) in higher cardiac vulnerability to I/R injury in late pregnancy. NP and LP(19 days of pregnancy Sprague-Dawley rats were subjected to 45 min myocardial ischemia followed by 3 hr reperfusion in vivo (I/R group). NP and LP rats which were not subjected to I/R injury served as controls. MicroRNA microarray expression were performed. In vitro, miR-98 was knocked down or overexpressed in neonatal rat ventricular myocytes(NRVMs). Forty eight hour post-transfection, the cells were subjected to 3 hr hypoxia followed by 12 hr reoxygenation. The apoptosis was detected by TUNEL staining, and the western blot was performed to validate the computational predicted targets genes, PGC-1α and STAT3. The miRNA expression profile showed that several miRNAs were differentially expressed in LP sham compared to NP sham, particularly, miR-98 which was significantly upregulated 1.7 fold in LP sham vs NP sham. Interestingly, miR-98 was upregulated even higher (2.3 folds) in LP rats when subjected to ischemia/reperfusion injury compared to NP I/R, suggesting that both ischemia reperfusion and late pregnancy regulate miR-98. Knock down of miRNA-98 in NRVMs significantly reduced apoptosis by ~50%, while over-expression of miR98 resulted in a two fold increase in apoptosis. In-vitro overexpression of miR98-KD in NRVMS resulted in the a significant upregulation of PGC-1α(normalized to scramble control, 1.34±0.04, P<0.05) and STAT3(normalized to scramble control, 1.28±0.05, P<0.05), whereas overexpression of miR-98 resulted in downregulation of both target genes(normalized to scramble control, 0.75±0.02 in PGC-1α; 0.5±0.03 in STAT3, both P<0.05). In conclusion, induction of miR-98 by pregnancy possibly through downregulating PGC-1α and STAT3 may at least in part underlie the greater cardiac vulnerability to I/R injury in late pregnancy.
Abstract 345: Inadequate Coupling between Ubiquitination and the Proteasome is a Major Pathogenic Factor of Myocardial Ischemia/Reperfusion Injury
Authors: Wang Xuejun , Hu Chengjun , Tian Yihao , Wang Hongmin , Wang Xuejun
Published: July 2021

Targeted removal of damaged/misfolded proteins is primarily done by the ubiquitin (Ub)-proteasome system (UPS). Generally, the UPS degrades a protein molecule via two main steps: ubiquitination of the protein and subsequent degradation of the ubiquitinated protein by the proteasome. The two steps are not always directly coupled. The proteasome can directly uptake nearby polyubiquitinated proteins via its Ub receptor subunits but extra-proteasomal Ub receptors may be required for shuttling the remote substrates to the proteasome for degradation. We have previously demonstrated that UPS function is impaired during acute myocardial ischemia/reperfusion (I/R) and this impairment plays a major role in I/R injury due to failure to timely remove damaged/misfolded proteins. The molecular underpinnings of UPS impairment in I/R injury remains poorly understood. This study shows an important role of Ubiquilin1, a
bona fide
extra-proteasomal Ub receptor, in cardiac ubiquitination-proteasome coupling during I/R. Mice with cardiomyocyte-restricted knockout of the Ubiquilin1 gene (CR-Ubqln1KO) or with Ubiquilin1 overexpression (Ubqln1OE) were subject to myocardial I/R created via left anterior descending artery ligation (for 30min) and subsequent release (for 24hr). The I/R induced decline of left ventricular maximum dP/dt, elevation of minimum dP/dt, and infarct size were significantly greater in CR-Ubqln1KO mice, and conversely, were markedly attenuated in Ubqln1OE mice, compared with their littermates with a respective control genotype. The accumulation of ubiquitinated proteins in I/R myocardium was increased by CR-Ubqln1KO and reduced by Ubqln1OE. Furthermore, in cultured cardiomyocytes, Ubqln1OE was able to enhance the degradation of a surrogate UPS substrate as well as a
bona fide
misfolded protein linked to human proteinopathy. These exciting new findings demonstrate for the first time that inadequate coupling between ubiquitination and proteasomal degradation hinders the degradation of damaged proteins in I/R myocardium and represents a major pathogenic factor to acute I/R injury. It is also suggested that facilitating the coupling is potentially a novel therapeutic strategy for reducing I/R injury.
Abstract 357: Intra-placental gene transfer of Insulin Like Growth Factor 1 Reprograms Altered Myocardial Gene Expression In Growth Restricted Mouse Model with Cardiac Dysfunction
Authors: omar khaled R , Alsaeid Tarek , Klanke Charles , Lababidi Ahmed , Habli Mounira
Published: July 2021

Background:
Intrauterine growth restriction (IUGR), defined as birth weight <10% for gestational age, is a risk factor for adult onset cardiovascular disease. We demonstrated that Intraplacental gene transfer of adenoviral insulin growth factor1 (Ad IGF1) rescues IUGR induced cardiac dysfunction in a surgical IUGR mouse model. The underlying molecular mechanism is unknown. We hypothesize that Ad IGF1 reprograms altered gene expression involved in collagen deposition in myocardium (Col1A1, CTGF, SRF, OPN, TGFβ)

Method:
Laparotomy was performed on pregnant C57 mice at embryonic day 18 and pups were divided into 3 groups. Control: Sham operated; IUGR: by ligation of a uterine artery branch; IUGR+IGF1: injection of Ad IGF1 after ligation. Pups were delivered and followed up with cross-sectional (2D) and Doppler transthoracic echocardiography until 36 week. Hearts were collected and RNA extracted for gene expression analysis by qPCR at 36 week. Data were analyzed using ANOVA

Result:
At week 36, IUGR mice showed significantly reduced EF (41 ± 6 vs 53 ± 7 vs 57 ± 9) compared to SHAM & IUGR+IGF1 (Fig A). IUGR showed significantly increased LVID;d (3.8 ± 0.3 vs 3.1± 0.49 vs 2.9 ± 0.4) as compared to SHAM & IUGR+IGF1 (Fig B). IUGR hearts demonstrated significantly increased CTGF (1.7± 0.6 vs 0.9 ± 0.3 vs 0.8 ± 0.2), and Col1A1 (2.4 ± 0.2 vs. 1.6 ± 0.4 vs 1.4 ± 0.5), as compared to SHAM & IUGR+IGF1(Fig C). No differences were detected in the other genes

Conclusion:
AdIGF1 gene transfer rescues IUGR induced cardiac dysfunction and reprograms myocardial gene expression of CTGF&COL1A1. These changes may represent a potential epigenetic regulation that reprograms the fetus and attenuates the risk of adult onset CVD
Abstract 40: Genetic Deletion Of Mir-208a Induces Pathological Remodeling And Heart Failure
Authors: Davis Robert T , Nagalingam Raghu S , Noor Mariam , Wang Da-Zhi , Wolska Beata M , Solaro R.John , Gupta Madhu
Published: July 2021

Introduction:
miR-208a, a small non-coding RNA expressed only in the heart, has a profound influence on cardiac gene expression. It has been previously demonstrated that genetic deletion of miR-208a does not affect viability or induce gross morphological heart defects, suggesting it is not required for normal cardiac growth and function (van Rooij et al., 2007; Callis et al., 2009). However, recent data from our lab indicate that miR-208a knock-out (KO) mice (developed in the laboratory of Da-Zhi Wang and subsequently maintained by inbreeding) display gross morphological cardiac abnormalities and dramatically reduced survival. Therefore, this investigation sought out to determine the mechanism(s) responsible for the maladaptive growth.

Methods:
Wild-type (WT) and miR-208a KO littermate mice (3-5 mon old) were used for this investigation. Cardiac function was assessed via echocardiography, cardiac fibrosis by collagen fiber staining, and gene expression by real-time PCR and Western blotting. We also measured basal Ca2+ transients and unloaded cell shortening in isolated mouse ventricular myocytes.

Results:
We observed KO mice have significantly lower survival rates, developed marked cardiac hypertrophy, fibrosis and have significantly reduced cardiac function. Compared to WT controls, cardiomyocytes from KO mice exhibited a significantly lower percent of cell shortening, slower rates of relaxation and contraction, lower amplitude, and slower kinetics (tau) of Ca2+ transients. Additionally, there was a reduced phosphorylation of phospholamban (Ser16) in miR-208 KO mice indicating a lower Ca2+ affinity of the SR Ca2+-ATPase.

Conclusions:
These data provide evidence that the observed reduction in cardiac function in miR-208a KO mice is likely due, in part, to alterations in cellular Ca2+ fluxes. To our knowledge, this is the first study to demonstrate genetic deletion of miR-208a induces a maladaptive phenotype at baseline. Our findings indicate the importance of considering variables such as genetic/environmental constraints when perturbing the expression of the miR-208a in the heart.
Abstract 43: Mice With Cardiac-Specific Inactivation Of Ppap2b (Lipid Phosphate Phosphatase 3) Validates Genome-Wide Association Studies.
Authors: Panchatcharam Manikandan , Miriyala Sumitra , Hendrix Natalie D , Escalante-Alcalde Diana
Published: July 2021

A recent meta-analysis of Genome-Wide Association Studies (GWAS) of coronary artery disease (CAD) involving over 86,000 individuals identified
PPAP2B
(encodes lipid phosphate phosphatase-3; LPP3) as a new loci (SNP rs17114036; P= 3.81 X 10
−19
) that independently predicts CAD. LPP3 is an integral membrane enzyme that dephosphorylates lysophosphatidic acid, sphingosine-1-phosphate and related bioactive lipids. Strikingly, we found that targeted inactivation of LPP3 in endothelial cells results in early embryonic lethality, in part to due to vascular patterning defects. In addition, we found that constitutive inactivation of LPP3 in the myocardium results in cardiac dysfunction, indicating that dysregulation of LPP3-dependent cardiomyocyte cell function. Heart rate was significantly higher in conditional
Ppap2b
Δ
mice (P<0.001), which may indicate a role for LPP3 in regulating heart rate and/or function. Further, we found that myocardial LPP3 levels are altered following myocardial infarction in mice and these results are in accord with our myocardial infarct data from patient that had down-regulated myocardial LPP3 levels. The major rs17114036A allele was associated with a 1.17 odds ratio for CAD. This SNP is located in the final intron of the six exon
PPAP2B
gene. At least seven SNPs are in robust linkage disequilbrium (r2>0.9) with rs17114036. The sequence surrounding the SNP rs17114036 matches a U1 spliceosome recognition sequence, and the variant base is located at the final position of a sequence with high homology to a U1 spliceosome 5’ spice site recognition motif. Binding of the U1 spliceosome may alter mRNA stability or processing, perhaps by masking cryptic splice sites, thus enabling efficient splicing or promoting polyadenylation of the mature message. To address the polymorphism, we transfected MDA MB 453 cells, which are homozygous for the “T’ allele of rs17114036 SNP, with a short synthetic 2’-O-methyl phosphorothioate RNA. We found a dramatic decrease in both LPP3 protein expression and LPP3 mRNA levels. These results imply that the region containing the rs17114036 SNP may be important for proper processing and/or stability of the LPP3 transcript thereby functionally validating the GWAS study.
Abstract 45: The Identification and Hierarchy of Bone Marrow-derived Artery-resident Mesodermal Progenitor Cells and their Dynamics in Atherosclerosis
Authors: Cho Hyun-Jai , Cho Hyun-Ju , Kwon Yoo-Wook , Park Young-Bae , Kim Hyo-Soo
Published: July 2021

Background:
We recently identified bone marrow (BM)-derived artery resident calcifying progenitor cells. Sca-1+PDGFRα- cells may possess bipotent (osteoblastic/osteoclastic) characteristics. However, the nature of progenitor cells remains elusive. Therefore, we investigated developmental hierarchy of progenitor cells and
in vivo
dynamics in atherosclerosis.

Methods and Results:
We harvested cells from BM and artery of C57 mice. In BM, Lin-CD29+Sca-1+PDGFRα- cells showed hematopoietic potential and differentiated into osteoclasts OC). They also possessed mesenchymal stem cell property including osteoblastic (OB) differentiation, suggesting that Sca-1+PDGFRα- cells could be mesodermal progenitor cells. Interestingly, BM-derived artery-resident, clonal Sca-1+PDGFRα- cells maintained bipotency (OB/OC) but lost hematopoietic nature. In contrast, Sca-1+PDGFRα+ cells in BM and artery only showed unipotency (OB). When we overexpressed or knocked down PDGFRα, there was no alteration in OB or OC differentiation of Sca-1+PDGFRα- cells and no effect on OB differentiation of Sca-1+PDGFRα+ cells, indicating PDGFRα as a surface marker but not a functional player. In hyperlipidemic ApoE-KO mice compared with control, Sca-1+PDGFRα- cells were less mobilized from BM to peripheral circulation and less infiltrated into atherosclerotic plaque, whereas Sca-1+PDGFRα+ cells were not significantly affected. Multiplex cytokine assay of serum and artery revealed that IL-1β was significantly increased and IL-5 was markedly decreased in atherosclerotic mice. IL-1β decreased the migration of Sca-1+PDGFRα- cells by 5 folds compared with TNFα, and IL-5 increased the migration as much as TNFα. But the migration of Sca-1+PDGFRα+ cells was not altered. These data indicate that atherosclerosis-related humoral factors mainly regulated mesodermal progenitor cells’ dynamics.

Conclusion:
We demonstrate that Sca-1+PDGFRα- cell is a mesodermal progenitor cell that possesses both hematopoietic and mesenchymal potentials. In atherogenesis, the mobilization and infiltration of Sca-1+PDGFRα- progenitor cells were regulated by IL-1β and IL-5. These data provide a novel mechanism regarding the role of bipotent progenitor cells in atherosclerosis.
Abstract 51: Aldosterone Induces Cardiac Deleterious Effects Through A Grk2 And Grk5 Dependent Pathway
Authors: Cannavo Alessandro , Liccardo Daniela , Bass Sarah M , Woodall Benjamin , Traynham Christopher J , Ibetti Jessica , Rengo Giuseppe , Koch Walter J
Published: July 2021

High Aldosterone (Aldo) plasma levels are present in heart failure patients and are associated with increased cardiac fibrosis and hypertrophy. These effects have been shown to be, in part, related to the transactivation of the angiotensin II receptor (AT1R), a G-protein coupled receptor (GPCR). In this regard, the GPCR kinase 2 (GRK2) and 5 (GRK5) are both critical regulators of cardiac function through GPCR regulation. Since these GRKs have also been shown to play a role in cardiac hypertrophy and HF development we investigated whether these kinases may play a role in myocardial Aldo signaling. To test this, we first treated neonatal rat ventricular cardiomyocytes (NRVMs) with Aldo (1 μM) and looked for GRK regulation. Following 12 hrs of Aldo-stimulation we observed a significant increase in both GRK2 and GRK5 protein levels. We also found that Aldo induced the localization of GRK2 to mitochondria, which the lab has previously found to occur following ischemic injury leading to increased cell death and mitochondrial dysfuntion. Indeed, high Aldo on NRVMs led to a significant increase in ROS generation and cell death, as observed by Mitosox Red and TUNEL staining, respectively. Notably, all these effects were abolished respectively by Spironolactone (Spiro, an Aldo receptor blocker) or Losartan (Los, an AT1R antagonist) pre-treatment. Next, in nuclear fractions, purified from Aldo-treated NRVMs, we observed a consistent increase in GRK5 nuclear localization that consequently induced a significant activation of hypertrophic genes, which is consistent with previous studies showing GRK5 being a key regulator of maladaptive cardiac hypertrophy after pressure-overload. Importantly, Spiro pre-treatment efficiently abolished these effects of GRK5. Finally, we treated wild-type mice in vivo with Aldo for two and four weeks and not only found that this induced significant cardiac hypertrophy and fibrosis compared to saline-treated mice, we also found significant increases in levels of GRK2 and GRK5. Therefore, our study provide, for the first time, data demonstrating that GRK2 and GRK5 are potential regulators of Aldo-mediated cardiac pathology acting either down-stream of mineralocorticoid receptor or transactivated AT1Rs.