bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 326: Cd36 Deficiency Reduces Obesity-associated Inflammation And Oxidative Stress In Heart
Authors: Hajri Tahar , Gharib Mohamed , Fungwe Thomas V
Published: July 2021

OBJECTIVE:
Obesity is often associated with diabetes and cardiovascular diseases (CVD). Mounting evidence shows that diabetes is associated with structural and functional changes in the heart. CD36 protein is highly expressed in heart and regulates lipid utilization in cardiacmyocytes. In this paper, we investigated the impact of CD36 expression on obesity-associated inflammation and oxidative stress in heart.

METHODS:
Studies were conducted in control lean (WT), obese leptin deficient (Lep
Ob/Ob
) and leptin deficient-CD36 null (Lep
Ob/Ob
-CD36
-/-
) mice. To examine obesity-associated insulin resistance, glucose uptake and insulin signaling were examined in adult mouse hearts. Presence of macrophages in heart was examined with immunohistochemisty. Oxidative stress makers and activity of anti-oxidant enzymes were measured in hearts. To evaluate substrate utilization, glucose and fatty acid oxidation was tested in primary cultures of ventricular myocytes. Finally, the activity of pro-inflammatory kinases p38 mitogen-activated protein kinases (p38-MAPK), c-Jun NH2-terminal kinases (JNK) were examined in cardiacmyocytes challenged with palmitate.

RESULTS:
In Lep
Ob/Ob
, glucose uptake and oxidation in heart was lower than lean WT mice, while cardiac FA oxidation was strongly higher. Silencing CD36 in Lep
Ob/Ob
mouse markedly improved insulin sensitivity and glucose uptake in heart, but resulted in marked reduction of FA oxidation. Immunostaining of heart sections with macrophage specific antibody F4/80 showed that macrophage content was higher in myocardium of Lep
Ob/Ob
mice than Lep
Ob/Ob
-CD36
-/-
mice. Moreover, oxidative stress markers, isoprostanes and reactive oxygen species, and expression of pro-inflammatory cytokines were higher in hearts of Lep
Ob/Ob
than Lep
Ob/Ob
-CD36-/- mice, although the activities of anti-oxidant enzymes were comparable. Chronic overload of Lep
Ob/Ob
cardiac myocyte with palmitate strongly induced the activity of JNK and p38-MAPK, but was less effective in Lep
Ob/Ob
-CD36
-/-
cardiac myocytes.

CONCLUSIONS:
These results show that CD36 deficiency induced a significant reduction of obesity-associated oxidative stress and inflammation in heart in parallel to a drop in fatty acid oxidation.
Abstract 327: Mitochondrial Functional Variation Contributes to Endothelial Cell Activation
Authors: Krzywanski David M , Cheng Bing , Shen Xinggui , Kevil Christopher
Published: July 2021

Vascular oxidant stress contributes to endothelial dysfunction and plays a critical role in early stage cardiovascular disease (CVD) development. Changes in endothelial function due to oxidant stress may contribute to CVD initiation and progression through the development of a pro-inflammatory environment. Differences in mitochondrial function may contribute to this process and provide insight into why age of onset and clinical outcomes differ amongst individuals form distinct ethnic groups; but no reports demonstrate distinct mitochondrial functional parameters between normal cells. Consequently, we hypothesized that significant variations in normal mitochondrial function and oxidant production exist between endothelial cells from donors representing different ethnic groups. Aspects of mitochondrial oxygen utilization and oxidant production were assessed under basal and inflammatory conditions in human aortic endothelial cells (HAECs) isolated from African Americans (AA) and Caucasians (CA). Bioenergetic analysis indicates that compared to CA, AA HAEC utilized significantly less oxygen for ATP production, possess a lower maximal respiratory capacity, and have reduced electron leak. Significant differences in mitochondrial membrane potential, decreased expression of endothelial nitric oxide synthase, and increased levels of superoxide were also observed and AA HAEC supporting a pro-inflammatory phenotype. As a marker of endothelial cell activation, AA HAEC expressed increased levels of intercellular cell adhesion molecule-1 under both basal and inflammatory conditions that could be partially mitigated but treatment with the mitochondrially targeted antioxidant MitoTEMPO. These data demonstrate that fundamental differences exist in mitochondrial oxygen utilization and oxidant production between CA and AA HAEC and that these changes may affect endothelial cell activation. These findings are consistent with the hypothesis that differences in “normal” mitochondrial function amongst ethnic groups could influence individual susceptibility by contributing to vascular inflammation, providing important insights into the mechanisms that contribute human CVD development.
Abstract 328: ES1 Is A Novel Mitochondrial Protein Protecting The Heart Via Direct Regulation Of Mitochondrial Energy Metabolism
Authors: Long Qinqiang , Yang Huan , Zhou Yiqun , Wang Aibing , He Lan , Li Yuquan , Kim Teayoun , Ding Yishu , Zhelyabovska Olga , Yang Qinglin
Published: July 2021

Defects in the myocardial energy metabolism have been linked to pathological cardiac hypertrophy and congestive heart failure. However, the regulation of myocardial energy metabolism remains obscure. ATP synthase is an enzyme complex in the mitochondria and plays a central role in energy metabolism. In this study, we identified ES1, a mitochondrial protein with unknown function, as a key determinant of myocardial energy metabolism via controlling ATP synthase activities. We uncovered that ES1 interacts with both α and β subunit of ATP synthase, and its expression levels in H9C2 cardiomyocytes were directly correlated to ATP synthesis and inversely to ATP hydrolysis. Cellular energetic analysis revealed that ES1 levels in H9C2 cardiomyocytes were directly correlated with mitochondrial oxidative metabolism. ATP synthase activity assays revealed increased synthesis activities and decreased hydrolysis activities on cardiac mitochondria from a mouse line with Cre-LoxP mediated, tamoxifen inducible, cardiomyocyte-restricted ES1 overexpression (TM-ES1oe) compared with mice of tamoxifen-inducible Mer-Cre-Mer (TMCM). We induced ES1 overexpression in TM-ES1oe mice (3-month-old) 7 days after transverse aortic constriction(TAC) and compared with TMCM mice with identical treatment. Echocardiography assessment revealed a substantially improved Ejection fraction (EF%) and Fractional shortening (FS%) and diminished left ventricular hypertrophy in TM-ES1oe mice compared with TMCM mice. Sections of TM-ES1oe hearts stained with Masson’s Trichrome blue showed markedly decreased interstitial fibrosis compared with TMCM control. We have also generated an ES1 knockout line. ES1 knockout mice(3-month-old)showed cardiac dysfunction with decreased EF% and FS% under a basal condition. Transmission electron microscope examination revealed substantial loss of mitochondrial cristae structure on ES1 knockout hearts. These results indicate that ES1 protecting the heart by direct regulation of mitochondrial energy metabolism. ES1 may be directly involved in pathological development of cardiac hypertrophy and heart failure. We suggest that ES1 is a potential therapeutic target in treating cardiomyopathy and other heart diseases.
Abstract 332: Loss of beta-catenin Leads to Induction of a Chondrogenic Phenotype in Adult Mouse Heart Aortic Valves
Authors: Fang Ming , Conway Simon J , Yutzey Katherine E
Published: July 2021

Objectives:
Altered Wnt/beta-catenin signaling has been implicated in human heart valve disease; however, a causal relation has not been established. This study is aimed at determining the role of Wnt/beta-catenin signaling in adult valve homeostasis and disease. In endochondral bone development, Wnt/beta-catenin signaling inhibits early chondrogenesis and promotes osteogenesis. Therefore, we hypothesize that loss of Wnt/beta-catenin signaling promotes chondrogenic differentiation of valvular interstitial cells (VICs) resembling myxomatous valve disease.

Methods & Results:
Periostin (Postn)Cre was used for conditional loss of beta-catenin (Ctnnb1) specifically in VICs in mice. PostnCre is active in valve mesenchymal progenitors by embryonic day (E)13.5, therefore bypassing requirements for beta-catenin in endothelial-to-mesenchymal transition and endocardial cushion formation. Histology of adult heart valves was analyzed by Masson’s Trichrome and Movat’s Pentachrome staining, and the expression of extracellular matrix (ECM) genes was examined at both mRNA and protein level. Heart valve development appears normal at both fetal and neonatal stages with loss of beta-catenin. However proteoglycan-rich nodules form in adult aortic valves (AoVs) lacking beta-catenin at 3 months with 100% penetrance. These nodular cells are enlarged and express nuclear Sox9 and chondrogenic ECM genes, including Aggrecan, Collagen type 2 (Col1) and Col10, reminiscent of hypertrophic cartilage. Fibrillar collagens 1 and 3, as well as Versican, are excluded from the nodules, accompanied by abnormal ECM deposition throughout the AoV leaflets. Likewise, treatment of cultured porcine aortic VICs with the Wnt pathway inhibitor XAV also induces Aggrecan and Col2 gene expression in vitro. This work demonstrates that beta-catenin is required for the maintenance of normal ECM deposition and composition in adult mice, and, therefore, is indispensible for adult heart valve homeostasis.

Conclusions:
We have demonstrated that beta-catenin is a key factor in maintaining normal valve ECM organization in adult AoV. In the absence of beta-catenin, VICs are susceptible to chondrogenic differentiation related to myxomatous valve disease.
Abstract 339: Heterozygous Disruption of Drp1 Enhances Myocardial Injury in Response to Ischemia/Reperfusion via Inhibition of Mitophagy
Authors: Ikeda Yoshiyuki , Zhai Peiyong , Ohishi Mitsuru , Sadoshima Junichi
Published: July 2021

Mitochondrial fission and fusion are essential for maintaining mitochondrial quality control. However, their role in stress resistance remains unknown. Since Dynamin-related protein 1 (Drp1) plays an essential role in mediating mitochondrial fission, we used cardiac-specific heterozygous Drp1 KO (Drp1-hetCKO) mice whose cardiac function is normal at 12 weeks of age. In order to evaluate the role of Drp1 in mediating stress resistance, 12-week-old Drp1-hetCKO and control (Ctr) mice were subjected to myocardial ischemia (30 min)/reperfusion (24 hours) (I/R). I/R injury increased the translocation of Drp1 from cytosol to mitochondria in Ctr mice, whereas this translocation was attenuated in Drp1-hetCKO mice.The infarct size/area at risk after I/R was significantly greater in Drp-1 hetero KO mice than in control mice (55.2 ± 3.0 vs. 40.2 ± 1.6%). Electron microscopic analyses showed that I/R significantly decreased mitochondrial mass and increased the number of autophagosomes containing mitochondria in control mice. However, these changes were significantly attenuated in Drp1-hetCKO mice, suggesting that endogenous Drp1 mediates mitochondrial fission and mitophagy after I/R (Relative mitochondrial mass, Ctr, baseline: 1.0±0.3, I/R: 0.7±0.4; Drp1-hetCKO, baseline: 1.8±0.3, I/R: 1.7±0.3). We also examined mitophagy using mitochondria-targeted Keima fluorescence. Keima has a bimodal excitation spectrum peaking at 440 and 560 nm, corresponding to the neutral and acidic pH states, respectively. The maturation of autophagosomes to autolysosomes can be monitored by measuring the fluorescence of mitochondria-targeted Keima. Fluorescent dots with high ratios of 560/440, indicating mitophagy, were significantly increased after GD in sh-scramble-transduced CMs (GD: 28.6±4.2, control 2.3±1.0 dots/cell, p<0.01) but not in sh-Drp1-transduced CMs (GD: 2.4±1.8, control 3.2±1.6 dots/cell, not significant), suggesting that GD stimulates mitophagy but that this is inhibited when Drp-1 is downregulated. These results suggest that endogenous Drp1 play an essential role in mediating mitochondrial fission and mitophagy, which in turn play an essential role in mediating myocardial protection in response to I/R.
Abstract 343: Activation of ROR-α, but not ROR-β or ROR-γ, Protects against Myocardial Ischemia/Reperfusion Injury
Authors: Pu Jun , He Ben , Gao Erhe , Ma Xinliang , Wang Yajing
Published: July 2021

Objectives:
The RAR-related orphan receptors (RORs) are members of the nuclear receptor superfamily that play a pivotal role in many physiological processes, including regulation of the circadian rhythm, development, metabolism and immune function. Three different but highly homologous ROR isoforms, ROR-α, -β, and -γ, have been discovered separately. However, the functional roles of RORs in the heart have never been investigated. We investigate the role of RORs in the pathophysiology of acute myocardial ischemia/ reperfusion (MI/R) injury.

Methods and Results:
The endogenous RORα, but not RORβ or RORγ, was significantly upregulated after MI/R. Synthetic ROR agonists SR1078 and SR3335 reduced myocardial infarction and improved contractile function after MI/R. Mechanistically, ROR activation inhibited endoplasmic reticulum (ER) stress, attenuated mitochondrial impairment, reduced cardiomyocyte apoptosis, and inhibited MI/R-induced autophagy dysfunction. Moreover, ROR activation inhibited MI/R-induced oxidative stress and nitrative stress. The aforementioned cardioprotective effects of ROR agonists were impaired in the setting of cardiac-specific gene silencing of RORα, but not RORβ or RORγ subtype. In contrast, adenovirus-mediated cardiac RORα overexpression, but not RORβ or RORγ overexpression, decreased myocardial infarct size and improved cardiac function through attenuating oxidative/nitrative stress and inhibiting ER stress, mitochondrial impairment, and autophagy dysfunction. Finally, RORα
sg/sg
mice (loss-of-function mutation in RORα), but not RORβ-null or RORγ-null mice, increased MI/R injury (greater apoptosis, larger infarct size, and poor cardiac function), exacerbated MI/R-induced oxidative/nitrative stress, and aggravated endoplasmic-reticulum stress, mitochondrial dysfunction, and autophagy dysfunction.

Conclusion:
Our study provides the first direct evidence that RORα acts as a novel endogenous cardioprotective receptor against myocardial injury. RORα, but not RORβ or RORγ, is a novel cardiac protective receptor against MI/R injury, supporting for the drug development strategies specifically targeting RORα for the treatment of ischemic heart disease.
Abstract 344: Basis for Bicarbonate Damage in Myocardial Ischemia/Reperfusion Injury
Authors: Queliconi Bruno B , Cunha Fernanda M , Gottlieb Roberta A , Kowaltowski Alicia J
Published: July 2021

In the clinical setting, bicarbonate is often used to correct acidosis arising from accumulated CO2/HCO3- during ischemia. We observed that HL-1 cardiomyocytes exposed to increased [CO2/HCO3-] had more cell death after hypoxia/reoxygenation (H/R) and Langendorff-perfused rat hearts had larger infarcts after ischemia/reperfusion (I/R). In addition to buffering pH, the CO2/HCO3- pair possess an underappreciated redox activity that may contribute to injury. In order to study the effects of high CO2/HCO3- during ischemic injury independent of pH, we clamped pH using HEPES and used the mouse cardiomyocyte HL-1 cell line and isolated perfused rat hearts. HL-1 cells exposed to 10% CO2/HCO3- had no damage under basal conditions but developed exaggerated protein carbonylation and cell death after H/R. In Langendorff-perfused rat hearts, 10% CO2 was well tolerated during baseline conditions but resulted in increased protein carbonylation, cell death and larger infarcts after I/R. We hypothesized that the increased oxidative damage to proteins could be due to mitochondrial dysfunction with greater ROS production, diminished proteasomal degradation of oxidized proteins, or impaired autophagic clearance of damaged mitochondria and oxidized protein aggregates. There was no differential effect of CO2 on mitochondrial morphology or proteasomal activity in HL-1 cells. In mitochondria isolated from perfused hearts subjected to I/R under low and high CO2 conditions, there was no difference in ROS production or oxidized protein content, suggesting mitochondrial damage was not affected by CO2 level. Examination of autophagy in HL-1 cells exposed to high CO2 during H/R revealed higher LC3-II and lower p62 content. In hearts, changes in LC3-II were inconsistent; however, we detected less p62 protein, less mitochondria-associated Beclin1, and significantly more LC3 mRNA in hearts exposed to 10% CO2 during I/R. Taken together, these findings suggest that 10% CO2 affects autophagy, which could explain the accumulation of oxidatively damaged proteins. These findings point to a protective role for autophagic clearance of oxidized protein aggregates during I/R injury that may be adversely impacted by bicarbonate therapy.
Abstract 350: High-Mobility Group Protein B2 is Essential in Maintaining Normal Cardiac Gene Expression
Authors: Chen Haodong , Rosa-Garrido Manuel , Gray Ricardo , Chen Zugen , Nelson Stanley F , Franklin Sarah , Monte Emma , Vondriska Thomas M
Published: July 2021

Heart hypertrophy is a complex disease that involves differential expression of hundreds of genes and requires highly coordinated chromatin remodeling events that must facilitate such genome-wide changes in gene expression. We examine the genome-wide distribution of a chromatin structural protein, High-Mobility Group Protein B2 (HMGB2) in isolated cardiac myocytes with or without adrenergic receptor agonist. We report a comprehensive map of HMGB2 binding in both normal and hypertrophic cardiac myocytes, and find that HMGB2 preferentially localizes to promoters, CpG islands, enhancers and transcription factor binding sites. Moreover, we find that upon hypertrophic stimulation, HMGB2 peaks move from regulatory elements to intergenic regions. Because both HMGB2 knock-down and PHE treatment can induce hypertrophic growth, we compare gene expression dynamics between HMGB2 knock-down and PHE treatment, and find hypertrophy-related gene expression pattern upon HMGB2 knock-down is different from the one after PHE treatment. This also indicates the existence of multiple pathways that lead to hypertrophic growth. Further study reveals that HMGB2 co-localizes with many other functional elements including p300 and CTCF. Interestingly, we find that HMGB2 binding profiles are different at the binding sites of four cardiac transcription factors, MEF2A, NKX2.5, GATA4 and SRF. More specifically, HMGB2 localization is higher at MEF2A binding sites, indicating that HMGB2 may be involved in facilitating the functioning of MEF2A. Finally, we calculate the DNA bendability score based on the primary sequence of genome and correlate it with HMGB2 binding intensity. Consistent with the understanding that HMGB2 bends DNA toward DNA major groove, we find a positive correlation between HMGB2 binding and DNA bending propensity. Surprisingly, PHE treatment in NRVMs induced the relocation of HMGB2 to less bendable intergenic regions. Taken together, our data conclude that HMGB2 is required for maintaining the normal cardiac gene expression pattern, and its binding to cardiac chromatin is dynamically altered during hypertrophic growth.
Abstract 352: Pulmonary Vein Thrombosis can make Pulmonary Vein Acidic States, which may be Associated with Atrial Fibrillation.
Authors: Takeuchi Hidekazu
Published: July 2021

Atrial fibrillation can cause ischemic stroke. To prevent atrial fibrillation (AF) is crucial to prevent ischemic stroke. The pulmonary vein has a myocardial layer that can generate spontaneous or triggered action potentials. The myocardial layer is extended from the left atrial myocardium. Pulmonary vein myocardium sleeve is known to be associated with generating and maintaining AF. Pulmonary vein myocardium can be classified into two types. One is short and thin myocardium sleeve, which has no potential to cause atrial fibrillation (AF). And the other is long and thick myocardium sleeve, which has potential to cause AF. The mechanisms of such myocardium sleeve changes are not understood well.

Pulmonary vein thrombosis (PVT) is believed to be rare, which was reported as a rare complication of chest surgeries such as lobectomy or lung cancers. But since 2012, I reported seven cases of PVT in elderly patients without such conditions, which suggests that PVT is not uncommon. That is a novel notion. PVT prevents arterial blood flow, which inhibits oxygen and nourishment supply and carbon dioxide excretion. Hypoxia activates hypoxia inducible factors (HIFs), and HIFs can modulate epigenetic changes, reprogramming and ES cells. Undernourishments may activate nuclear respiratory factor-1 (NRF-1) and the aryl hydrocarbon receptor (AhR). PVT can make pulmonary vein acidic states by inhibiting excretion of carbon dioxide and may modulate pulmonary vein myocardium. Under acidic states, pulmonary vein cells such as pulmonary vein myocardium cells may obtain some kinds of multipotency. After obtaining multipotency, the cells may turn into new cells to adapt changed surroundings. The changes of myocardium sleeve may be caused by acidic state conditions and HIFs, AhR and NRF-1, which seemed to modulate pulmonary vein myocardium functions. To clear these, more studies are needed.
Abstract 354: Haploinsufficiency Of Sam68 Attenuates Vascular Inflammation And Atherosclerosis In Apoe-deficient Mice
Authors: Han Shuling , Zhou Junlan , Qin Gangjian
Published: July 2021

Background:
Chronic inflammation in the arterial wall is a major drive of atherosclerosis and is largely mediated by the NF-kappaB signaling pathway in the vascular and pro-inflammatory cells, notably macrophages. Recently, Sam68, an adaptor protein and Src kinase substrate, has been shown to promote NF-kappaB signaling in mouse embryonic fibroblasts (MEFs). However, whether Sam68 plays a role in the development of atherosclerosis is currently unknown.

Methods and Results:
To evaluate the role of Sam68 in atherosclerosis, we crossed Sam68(+/-)mice (Sam68-/- mice are sterile) with ApoE(-/-)mice and obtained ApoE(-/-)Sam68(+/-)and ApoE(-/-)sam68(+/+)mice, which are both grossly normal. Fed on a regular chow, time-course analyses of atherosclerotic lesions were performed histologically throughout the aortas in these mice. ApoE(-/-)Sam68(+/-) mice displayed a significantly delayed development of atheromatous plaques as compared to ApoE(-/-)sam68(+/+)mice at 5, 7, and 10 months of age. The expression levels of pro-inflammatory factors, including TNFalpha, IL-1beta, and IL-6, in the aortas were significantly lower in ApoE(-/-)Sam68(+/-)mice than in ApoE(-/-)Sam68(+/+) mice. Consistently, in the cultured Raw264.7 macrophages, knockdown of Sam68 resulted in a significant reduction in the TNFalpha-induced expression of these pro-inflammatory genes and also in the level of nuclear phospho-p65, indicating an attenuated NF-kappaB activity. Similar results were reproduced in primary macrophages that were isolated from peritoneum or differentiated from bone-marrow mononuclear cells of Sam68(-/-) mice vs. wild-type mice.

Conclusions:
Our results for the first time suggest that Sam68 is a critical component of the pro-inflammatory NF-kappaB signaling pathway in macrophages and modulates the course of atherosclerosis at least in ApoE(-/-) mice. Additionally, we have performed Sam68 co-immunoprecipitation and identified by mass spectrometry a panel of new Sam68-interacting proteins. Further experiments are under way to validate those Sam68-interacting proteins that contribute to the NF-kappaB signaling pathway.
Abstract 355: Post-translational Modification of Histone Variants in Cardiac Hypertrophy
Authors: Miller Mickey , Makaju Aman , Wang Li , Franklin Sarah
Published: July 2021

While global changes in gene expression are a hallmark of cardiac hypertrophy, much less is known regarding the epigenetic factors driving these changes. Local chromatin packing and gene accessibility, which governs transcriptional status, has been correlated with specific post-translational modifications on the histone tails of nucleosomes occupying these regions. However, the specific alterations in histone post-translational modifications driving gene expression changes during cardiac hypertrophy are largely unknown. To identify myocyte specific changes in histone post-translational modifications during cardiac hypertrophy we performed label-free quantitation of nuclear proteins from isolated neonatal rat ventricular myocytes exposed to the hypertrophic agonists, phenylephrine and isoproterenol. Peptide samples were analyzed on a Thermo Orbitrap Velos Pro mass spectrometer using CID & HCD fragmentation. Differential expression analysis was performed using the Progenesis LC-MS software where modified histone peptides were normalized against total protein expression. We observed multiple known and novel post-translational modifications on each of the four core histones, many of which changed in the setting of hypertrophy. To validate these findings in an animal model we performed the same analysis of histone post-translational modifications from cardiac tissue of mice under basal conditions or after pressure-overload induced hypertrophy. This study provides the first global characterization of myocyte specific changes in histone post-translational modifications in cardiac hypertrophy and highlight basic mechanisms of genomic reprogramming operative in disease.
Abstract 358: Neonatal Mouse Heart Maturation: Transcriptome-Wide Analysis Reveals Chamber-Specific Exon Enrichment of Cell Cycle Genes
Authors: Touma Marlin , Kang Xuedong , Lee Jae-Hyung , Xiao Xinshu , Wang Yibin
Published: July 2021

Background:
During postnatal maturation of mammalian heart the neonatal cardiomyocytes undergo dramatic changes including complete maturation, proliferation arrest, and terminal exit from the cell cycle (CC). However, transcriptome-wide analysis of CC programs has not been performed in perinatal stages among different cardiac chambers. In particular, the contribution of alternative RNA splicing to the chamber-specific CC activities is unexplored

Design/Methods:
To achieve comprehensive analysis of differential expression (DE) and alternative splicing (AS) of CC-related genes in left ventricle (LV) versus right ventricle (RV) during maturation deep RNA-seq was performed on male newborn mouse LV and RV at 3 time points of perinatal transition: P0, P3 and P7. Reads were mapped to mouse Transcriptome, and to mouse Genome. Transcriptome-Wide difference in inclusion of individual exons was performed using MATS. DE genes and AS variants were defined as those with fold change ≥2, at expression level ≥3 RBKM and a false discovery rate ≤0.05. Significant gene ontology (GO) terms were determined at P-value ≤0.05. Levels of expression were validated using qRT-PCR

Results:
Altogether, 2116 DE genes and 1162 AS events were observed. Among them, 109 CC-related genes were further analyzed. Distinct temporal patterns of DE and GO enrichment of CC genes in LV vs. RV during maturation were identified. Chamber -specific induction of genes involved in mitosis, karyokinesis, and cytokinesis was found at P7. RNA Splicing analysis of CC genes revealed 77 AS events. Skipping exon accounted for nearly half splicing events. Among 30 spliced exon variants, significant chamber-and temporal-specific inclusion were observed. Interestingly, the majority of AS variants exhibited opposing patterns of exon usage in RV vs. LV at p7

Conclusions:
Our findings suggest novel molecular basis for chamber-specific programming of cellular proliferation and maturation in neonatal heart, including potential splicing regulation of dynamic exon enrichment of cell cycle related genes. Further functional studies to decipher putative splicing regulators of CC programming in LV vs. RV during maturation will likely lead to novel chamber-specific regenerative and therapeutic targets.
Abstract 37: The Positive Inotropic S100a1 Prevents Arrhythmogenic Sarcoplasmic Reticulum Ca2+ Leak And Ventricular Arrhythmias
Authors: Ritterhoff Julia , Völkers Mirko , Seitz Andreas , Spaich Kristin , Peppel Karten , Pleger Sven , Zimmermann Wolfram , Friedrich Oliver , Fink Rainer , Koch Walter , Katus Hugo , Most Patrick
Published: July 2021

S100A1 has emerged as a key factor in the control of cardiomyocyte (CM) contractile performance. Improved sarcoplasmic reticulum (SR) function with enhanced Ca2+ resequestration appears critical for its cAMP-independent inotropic effects but raises concerns about potential diastolic SR Ca2+ leakage that might trigger fatal arrhythmias. Thus, the goal of this study was to determine the impact of S100A1 on ryanodine receptor 2 (RyR2)-mediated SR Ca2+ leakage in vitro and in vivo.

S100A1 association with the RyR2 was significantly diminished (-50%) in failing cardiomyocytes and hearts with S100A1 downregulation, as shown by co-immunofluorescence, co-immunoprecipitation and proximity ligation assay. Adenoviral-mediated S100A1 overexpression (3-4 fold vs. GFP-control) in quiescent NCs (normal CMs) and FCs (failing CMs) decreased SR Ca2+-frequency (-50 and −40% respectively) and protected from β-AR-triggered diastolic Ca2+-waves (-62 and −58% respectively) in electrically stimulated (2Hz) CMs as assessed by epifluorescent and confocal Ca2+ imaging. In multicellular rat engineered heart tissue (EHT), S100A1-overexpression (6-8 fold vs. GFP-control) protected from Ca2+-triggered after-contractions (ACs) (-50%) with preserved enhancement of isometric twitch force (TF, +40%) at 2Hz. S100A1-mediated rescue of contractile failure of endothelin-1-treated EHT (-50% decrease in TF) was associated with protection from Ca2+-triggered ACs. In mice with post-ischemic heart failure, AAV9-mediated therapeutic administration of S100A1 enhanced S100A1/RyR2 association and prevented epinephrine-induced VTs (70% in MI group vs. 30% in MI-S100A1 group). Mechanistically, S100A1-overexpression changed neither PKA/CaMKII RyR2 phosphorylation pattern nor binding of accessory proteins like FKBP12.6, calmodulin or sorcin to RyR2 but enhanced S100A1/RyR2 stoichiometry.

Our data provide evidence that S100A1 interaction with the RyR2 can beneficially modulate and reverse diastolic RyR2 function dysfunction. S100A1 appears to convey a rather unique molecular profile combining cAMP-independent inotropy with protection against Ca2+-triggered arrhythmias.
Abstract 44: Interaction Of Osr1 And Tbx5 Is Involved In The Mouse Limb And Heart Development
Authors: Xie Linglin , Zhou Lun , Olson Patrick , O’Keefe Jacy , Fu Qiang
Published: July 2021

Mutations of TBX5 cause Holt-Oram syndrome (HOS) in human, a disease characterized by upper limb and heart defects. Mouse embryos of
Osr1
knockout caused similar heart defects, while the upper limb defects have never been reported. By genetically marking
Osr1
expressing cells in mice, using
Osr1:CreERT2
, we showed that
Osr1
expression cells contribute to the atrial septum progenitors between E8.0 and E11.0, and to the forelimb after E9.0. The expression of
Osr1
in the forelimb showed a gradient decreasing pattern from the digit 5 to digit 1. Conditional-
Tbx5
haploinsuffiency, using
Osr1:CreERT2
, compound with
Osr1
haploinsuffiency induced more incidence of atrial septal defects (ASDs) and double outlet right ventricle (DORV). Forty percent of these embryos also had digit defects: the digits are either missing, fused or lack normal identity, which were not observed in mouse embryos of either
Osr1
or
Tbx5
haploinsuffiency. Detailed study of the cardiac progenitors of the compound haploinsufficinecy for Tbx5 and Osr1 showed decreased proliferation in the posterior second heart field, which was associated with lower number of cells transiting from G2 to M phase and less gene expression of
Cdk6
and
CyclinD2
. In summary, our study demonstrated that interaction of
Osr1
and
Tbx5
is involved in the mouse limb and heart development and provides a potential mechanism for HOS.
Abstract 46: Temporal Ubiquitin-Proteasomal Degradation of Smad9 Mediated by its Specific E3 Ligase Asb2 is Required for Cardiac Development through the Regulation of Tbx2 Expression
Authors: Min Kyung-Duk , Asakura Masanori , Ito Shin , Imazu Miki , Shindo Kazuhiro , Kitakaze Masafumi
Published: July 2021

Background:
The bone morphogenetic protein (BMP) pathway plays crucial roles in cardiac development. Recent studies have reported that mutations in Smad9, one of the regulatory Smad specific for the BMP pathway, might result in cardiovascular diseases. However, both regulation and function of Smad9 in the cardiovascular system have not been elucidated.

Methods and Results:
We conducted DNA microarray using P19CL6 cells with forced expression of Smad9. Microarray analysis using Ingenuity Pathway Analysis elucidated that 19 genes including Tbx2 were related to BMP pathway and showed significantly altered expression levels by transient expression of Smad9. We confirmed by qRT-PCR that only Tbx2, but not other Tbx families, were induced by Smad9. Importantly, the expression of Tbx2 was more up-regulated by Smad9 than by Smad1. Moreover, we identified Asb2 as a specific E3 ligase that targets Smad9, but not Smad1/5, for proteasomal degradation. The in situ hybridization using murine embryo revealed that Asb2 is expressed predominantly in the heart during embryonic development, suggesting that Asb2 quantitatively regulates Smad9 in the developing heart. Biochemical analysis demonstrated that Tbx2 expression induced by Smad9 was attenuated by Asb2, which was restored by the treatment with proteasome inhibitor, lactacystin. Developmental studies using both P19CL6 cells and zebrafish showed that the ablation of Asb2 leads accumulation of Smad9 resulting in the up-regulation of Tbx2, which attenuates myocardial development while induces non-myocardial tissue including cardiac cushion. Indeed, alcian blue staining of morpholino-mediated knockdown of zebrafish Asb2 showed significantly dilated ventricle and thinned ventricular wall, accompanied with decreased myocardium and increased cardiac jelly.

Conclusions:
Smad9 induces the expression of Tbx2 during cardiac development and is temporally and quantitatively regulated by its specific E3 ligase Asb2. This is the first study to show both the target gene and specific E3 ligase of Smad9.
Abstract 48: Age-associated Imbalance of Vasorin/TGF-β1 Signaling in VSMC Facilitates Collagen Production
Authors: Wang Mingyi , Pintus Gianfranco , Giordo Roberta , Zhang Jing , Jiang Liqun , Monticone Robert , Lakatta Edward
Published: July 2021

Collagen deposition, a hallmark of arterial aging that resembles post-injury arterial restenosis, is perpetrated by angiotensin II (Ang II) signaling in arterial wall. Collagen aggregation at sites of arterial injury is regulated by the coordinated signaling of pro-fibrotic TGF-β1 and anti-fibrotic vasorin within VSMCs. The Ang II/TGF-β1/vasorin signaling relationship within VSMCs with aging, however, remains unknown. In vivo studies in old vs. young FXBN rats show that aortic transcription and translation of vasorin markedly decrease with aging. In vitro studies in VSMCs isolated from old vs. young aortae. Ang II-associated reduction of vasorin protein abundance in young VSMCs and age-associated changes in vasorin protein levels are reversed by the AT1 antagonist, Losartan (Los) (Figure). Dual immunolabeling and co-immunoprecipitation demonstrate that the co-incidence and physical interaction of vasorin and TGF-β1 within VSMCs are significantly decreased with aging. Importantly, exposure of young VSMCs to Ang II that increases p-SMAD2/3 and collagen type I production, mimicking old cells, and this effect is abolished or substantially mitigated by Los treatment, overexpression of ectopic vasorin, or exogenous recombinant human-vasorin protein. In contrast, exposure of old VSMCs to Los decreases p-SMAD2/3 and collagen type I production.Thus, an imbalance of the Ang II/TGF-β1/vasorin signaling cascade, a feature of the aged arterial wall, enhances the collagen production by VSMCs. Maintaining this signaling balance is a novel measure to retard adverse extracellular matrix remodeling, a determinant of arterial stiffening with aging. (MW and GP co-first authors)
Abstract 5: Neovascularization By Substance-p- Mobilized Epc And Msc In Vitro And In Vivo
Authors: Hong Hyun Sook , Kim Suna , Son Youngsook
Published: July 2021

Bone marrow stem cells, especially, endothelial precursor cells (EPC), mesenchymal stem cells (MSC) or hematopoietic stem cell (HSC) are expected as reparative cells for the repair of a variety of tissue damages such as stroke and myocardial infarction, even though their role in the repair is not demonstrated. This report was investigated to find a role of Substance-p (SP) as a reparative agent in the tissue repair requiring EPC and MSC.

In order to examine EPC (EPC
SP
) and MSC (MSC
SP
) mobilized by SP, we injected SP intravenously for consecutive 2 days and saline was injected as a vehicle. At 3 post injection, peripheral blood (PB) was collected.To get mesenchymal stem cells or endothelial progenitor cells, MNCs were incubated in MSCGM or EGM-2 respectively for 10 days.

Functional characteristics of the EPC
SP
were proven by the capacity to form endothelial tubule network in the matrigel in vitro and in the matrigel plug assay in vivo. In contrast, MSC
SP
did not form a tube-like structure but formed a pellet-structure on matrigel. However, when both cells were premixed before the matrigel assay, much longer and branched tubular network was formed, in which a-SMA expressing MSC
SP
were decorating outside of the endothelial tube, especially enriched at the bifurcating point. MSC
SP
may contribute and reinforce elaborate vascular network formation in vivo by working as pericyte-like cells. Thus, the EPC
SP
and MSC
SP
were labeled with PKH green and PKH red respectively and their tubular network was examined. Well organized tubular network was formed, which was covered by PKH green labeled cells and was decorated in a punctate pattern by PKH red labeled cells. In order to investigate the role of EPC
SP
and MSC
SP
specifically in vivo, rabbit EPC
SP
and MSC
SP
were transplanted to full thickness skin wound. The vessel of EPC
SP
-transplanted groups was UEA-lectin+, which was not covered with a-SMA+ pericytes but EPC
SP
+ MSC
SP
-transplanted groups showed, in part, a-SMA+ pericyte-encircled UEA-lectin+ vessels. This proved the specific role of MSC
SP
as pericytes.

From these data, we have postulated that the collaboration of MSC and EPC is essential for normal vessel structure and furthermore, accelerated wound healing as ischemia diseases, which can be stimulated through by SP injection.
Abstract 50: Therapeutic Silencing of miRNA-652 Restores Cardiac Function and Attenuates Pathological Remodeling in a Mouse Model of Pressure Overload
Authors: Bernardo Bianca C , Nguyen Sally S , Winbanks Catherine E , Gao Xiao-Ming , Boey Esther J , Tham Yow Keat , Kiriazis Helen , Thomas Colleen J , Lin Ruby C , Du Xiao-Jun , McMullen Julie R
Published: July 2021

Introduction:
Targeting microRNAs differentially regulated in settings of stress and protection could represent a new approach for the treatment of heart failure. miR-652 expression increased in hearts of a cardiac stress mouse model and was downregulated in a model of cardiac protection.

Aim:
To assess the therapeutic potential of silencing miR-652 in a mouse model with established pathological hypertrophy and cardiac dysfunction due to pressure overload.

Methods:
Mice were subjected to a sham operation (n=10) or transverse aortic constriction (TAC, n=14) for 4 weeks to induce hypertrophy and cardiac dysfunction. Mice were subcutaneously administered a locked nucleic acid (LNA)-antimiR-652 or LNA-control. Cardiac function was assessed by echocardiography before and 8 weeks post treatment, followed by molecular and histological analyses.

Results:
Expression of miR-652 increased in hearts subjected to pressure overload compared to sham operated mice (2.9 fold, n=3-5, P<0.05), but was silenced in hearts of mice administered LNA-antimiR-652 (95% decrease, n=3-7, P<0.05). In mice subjected to pressure overload, inhibition of miR-652 improved cardiac function (29±1% at 4 weeks post TAC compared to 35±1% post treatment, n=7, P<0.001) and attenuated cardiac hypertrophy. Functional and morphologic improvements in hearts of treated mice were associated with reduced cardiac fibrosis, apoptosis, cardiomyocyte size; decreased B-type natriuretic peptide gene expression; and preserved angiogenesis (all P<0.05, n=4-7/group). Mechanistically, we identified Jagged1, a Notch1 ligand, as a direct target of miR-652 by luciferase assay. Jagged1 and Notch1 mRNA were upregulated in hearts of TAC treated mice (1.2-1.7 fold, n=7, P<0.05). Importantly, chronic knockdown of miR-652 was not associated with any notable toxicity in other tissues.

Conclusion:
Therapeutic silencing of miR-652 protects the heart against pathological cardiac remodeling and improves heart function via mechanisms that are associated with preserved angiogenesis, decreased fibrosis and upregulation of a miR-652 target, Jagged1. These studies provide the first evidence that targeted inhibition of miR-652 could represent an attractive approach for the treatment of heart failure.
Abstract 7: Intracoronary Autologous Cardiac Progenitor Cell Transfer In Children With Hypoplastic Left Heart Syndrome: 2-year Results Of The Ticap Trial
Authors: Ishigami Shuta , Tarui Suguru , Okuyama Michihiro , Ousaka Daiki , Kobayashi Junko , Kasahara Shingo , Sano Shunji , Oh Hidemasa
Published: July 2021

Backgrounds:
Hypoplastic left heart syndrome (HLHS) is a severe congenital heart malformation.

Objective:
The aim of this study is to determine whether intracoronary delivery of autologous cardiosphere-derived cells (CDCs) was feasible and safe to treat the children with HLHS.

Methods:
Four-teen patients with HLHS undergoing staged palliations were prospectively enrolled in this trial between January, 2011, and January, 2012. Seven patients constitutively assigned to receive intracoronary CDCs injection followed by 7 patients allocated to a control group with standard care alone. The primary endpoint was to assess the safety and the secondary endpoint was the preliminary efficacy by assessing the improvements of the right ventricular function during the follow-up.

Results:
No major complications were reported within 24 months of CDC infusion. Echocardiography showed that improvement of right ventricular ejection fraction (RVEF) was greater in the CDC-treated group (+5.3±3.2%) than in controls (+0.1±3.4%, P=0.01) at 3-month follow up. This cardiac function enhancement was manifested even in long-term observation (+7.8±4.9% vs. +2.2±3.1% at 1 year, P=0.03; +8.8±3.7% vs. +3.4±6.4% at 2 years, P=0.04). The absolute improvements in RVEF between 2 groups was confirmed by using right ventriculogram (RVG: +8.9±7.6% vs. +2.0±2.8% at 1 year, P=0.02; +8.1±6.0% vs. +2.9±3.9% at 2 years, P=0.04). In addition, RVEF on cMRI was also markedly improved in CDC-treated patients from 36.1±7.5% at baseline to 42.7±8.7% at 1 year (P=0.04) and to 42.4±7.6% at 2 years (P=0.047). Heart failure status was reduced in CDC-treated group as shown by significant decrease in Ross Heart Failure Class (2.6±0.8 at baseline vs. 1.4±0.5 at 2 years, P=0.01). Moreover, Z scores for weight-for-age was significantly increased from −4.0±2.7 at baseline to −2.2±1.4 at 2 year (P=0.02), whereas all of these parameters did not change in control subjects.

Conclusion:
These results of 2-year follow-up of TICAP trial suggest that intracoronary infusion of autologous CDCs is feasible and safe to treat the children with HLHS. This novel therapeutic strategy may impact on cardiac function as well as clinical symptom of heart failure status and somatic growth in long-term outcome.
Abstract 71: A Dual PI3K-mTOR Inhibitor Induced Hyperglycemia and Increased Insulin Signals in the Heart
Authors: Yan Xinhua , Onufrak Jillian , Yang Yongyao , Lee Juyong , Fuseler John , Sun Maoyun , Sasi Sharath , Goukassian David , Hlatky Lynn , Price Robert L , Carrozza Joseph , Borg Thomas K , Morgan James P
Published: July 2021

We previously reported that a dual PI3K-mTOR inhibitor BEZ235 (BEZ) induced cardiac hypertrophy. Here, we investigated potential mechanisms.

Methods:
three month old FVB/n female mice were treated with BEZ for five weeks. Cardiac function was monitored by serial echocardiography during the treatment and hemodynamic measurements at the end of the study. Cell signaling was analyzed by RT-PCR, Western blotting and ELISA.

Results:
BEZ induced a dose-dependent increase of left ventricular (LV) wall thickness and systolic function. These were associated with increased hypertrophic markers ANP, BNP, β-MHC and α-skeletal actin in the heart. In addition, in chronic BEZ-treated mouse hearts, the activations of PI3Ks, mTOR and ERK were increased. We conducted further studies to understand these contradictory results. We found that BEZ induced an increase of hepatic gluconeogenesis gene expression which was associated with increased fasting glucose, increased serum insulin level, a worsened glucose and pyruvate tolerance and increased IGFR/Insulin receptor activation in the heart. Injections of insulin lowered blood glucose, improved glucose and pyruvate tolerance, but further aggravated BEZ-induced cardiac dysfunction. On the other hand, OSI-906 (an IGFR/IR inhibitor) normalized cardiac function in BEZ-treated mice.

Conclusions:
Chronic BEZ treatment induced cardiac hypertrophy may be caused by increased insulin receptor activation in the heart.
Abstract 83: β1 Vs β2 Adrenergic Signaling Differentially Regulates Mitochondrial Dynamics Through Alterations In Calcineurin And Drp1
Authors: Coronado Michael , Fajardo Giovanni , Zhao Mingming , Hu Dongquing , Stotland Aleksandr , Reddy Sushma , Gottlieb Roberta , Bernstein Daniel
Published: July 2021

Signal transduction through β1 and β2-adrenergic receptors (ARs) is considered a primary mechanism for regulating cardiovascular function and remodeling. Upon β-AR activation (i.e., physical activity, cardiac pathology) inotropy and chonotropy increase and mitochondria must quickly meet increased energy demand. This suggests that βARs and mitochondria are coupled mechanistically to rapidly respond to the functional and energetic needs of the heart. To investigate the role of β1 vs. β2-AR signaling on mitochondrial dynamics, we compared β1-/- and β2-/- to WT controls. β2-/- had increased mitochondrial fragmentation (increased number and decreased size) by electron microscopy vs. both WT and β1-/-. β2-/- showed altered regulation of mitochondrial fission: increased Drp1 translocation to the mitochondria vs. WT, whereas β1-/- had lower Drp1 translocation. These data suggest differential regulation of fission by βAR signaling, β1 activating and β2 suppressing fission. Since Ca2+-dependent calcineurin is known to activate Drp1 and [Ca2+]i is differentially regulated by β-AR signaling, we examined calcineurin as the bridge between β-AR signaling and Drp1 activation. In β2-/-, both Ca2+ transients and calcineurin activity were increased, suggesting β1-AR/Ca2+/calcinurin-mediated fission. To quantify mitochondrial fragmentation and biogenesis, mitotimer-transfected C2C12 cells were treated with the non-specific β-AR agonist isoproterenol resulting in mitochondrial fragmentation that was inhibited by the β1-antagonist CGP 12177 but not by the ß2-antagonist ICI 118551. Taken together, our data indicate that β1 and β2-AR signaling differentially regulate mitochondrial dynamics in the heart through alterations in [Ca2+]i, leading to calcineurin-induced translocation of Drp1.