bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4: Lymphatic Vessels Mediate the Mobilization of Cardiac Progenitor Cells after Myocardial Infarction
Authors: Goichberg Polina , Cimini Maria , Cannata Antonio , Signore Sergio , Waight Kanako , Rota Marcello , Anversa Piero , Leri Annarosa
Published: July 2021

The delivery of adult cardiac progenitor cells (CPCs) or their activation in situ constitute an evolving approach for the treatment of heart failure. CPCs are endowed with regenerative capacity, producing differentiating myocytes and vascular structures in the course of homeostasis and upon injury. The regenerative function of CPCs is contingent to their ability to migrate to and engraft within the wounded area. Yet, the mechanisms governing CPC trafficking in the diseased myocardium are largely unknown. The lymphatic system is vital for tissue repair, and the role of the lymphatic vasculature in the trafficking of hematopoietic and cancer cells is well documented. We examined whether cardiac lymphatic vessels mediate the translocation of CPCs in the infarcted myocardium. By imaging of the heart from transgenic c-kit-GFP reporter mice, we found that as early as 4 hours after myocardial infarction (MI), uncommitted lineage-negative progenitors accumulated in the vicinity of the lymphatic vessels located in the region bordering the necrotic area. Histologically, extensive lymphangiogenesis was documented in the mouse heart in the acute (8-48 hours) and chronic (15-35 days) phases of infarct healing and scar formation. CPCs were detected traversing the wall of lymphatic vessels at different stages after MI, indicative of the functional role of the lymphatic circulation in the recruitment of primitive cells to the site of injury. Furthermore, isolated human CPCs exhibited chemotaxis and specific binding to the human lymphatic endothelial cells (LECs) in steady-state conditions and, increasingly, after exposure to an inflammatory cytokine, TNFα. CPCs performed trans-endothelial migration in vitro, and actively intravasated into the lumen of microvessels formed by LECs in three-dimensional matrices. Finally, our data suggest that sphingosine-1-phosphate (S1P)-stimulated signaling governs the interactions of CPCs with LECs. These findings on the direct role of lymphatic vasculature in CPC trafficking may contribute to the development of novel therapeutic modalities to increase mobilization of endogenous or transplanted CPCs, promoting myocardial repair in patients with ischemic heart diseases.
Abstract 42: Differential Contribution Of The N- And C-terminal Domains To The Folding And Function Of Tandem Calponin-homology Domains
Authors: Mallela Krishna , Bandi Swati , Singh Surinder , Armstrong Geoffrey
Published: July 2021

Tandem calponin-homology (CH) domains constitute a major class of actin-binding domains that include dystrophin and utrophin, the two key proteins involved in muscular dystrophy. Despite their importance, how their structure controls their function is not understood. Here, we study the contribution of individual CH domains to the actin-binding function and thermodynamic stability of utrophin’s tandem CH domain. Traditional actin co-sedimentation assays indicate that the isolated C-terminal CH2 domain binds weakly to F-actin when compared with the full-length tandem CH domain. In contrast, isolated CH1 binds to F-actin with a similar efficiency as that of the full-length tandem CH domain. Thus, the obvious question that arises is why tandem CH domains require CH2, when their actin-binding efficiency is originating primarily from CH1. To answer, we probed the thermodynamic stabilities of individual CH domains. Isolated CH1 domain is unstable and is prone to serious aggregation. Isolated CH2 is very stable, even appears to be more stable than the full-length tandem CH domain. In addition, the CH2 domain, which is more stable, is less functional. These results indicate that the main function of CH2 is to stabilize CH1. Consistently, the proposed structure of utrophin’s tandem CH domain based on earlier X-ray studies indicates a close proximity between the C-terminal helix of CH2 and the N-terminal helix of CH1, and this helix in CH2 is more dynamic in the full-length protein when compared with that in the absence of CH1, suggesting the mechanism by which CH2 stabilizes CH1. These observations indicate that the two CH domains contribute differentially to the folding and function of tandem CH domains, although both domains essentially have the same native structure in the tandem CH domain. The N-terminal domain determines the function, whereas the C-terminal domain determines the stability.

This work was funded by the AHA Grant 11SDG4880046.
Abstract 53: Pathogenic role of DNA Single Strand Break Accumulation in Heart Failure
Authors: Higo Tomoaki , Naito Atsuhiko , Sakata Yasushi , Komuro Issei
Published: July 2021

Various environmental stress including reactive oxygen species (ROS) causes nuclear DNA damage. Increased production of ROS is observed in the failing heart and is considered as one of the causes of heart failure. Accumulating evidences suggest the presence of DNA damage in the failing heart, however, mechanistic link that connects DNA damage and heart failure remains elusive. Here, we show that DNA single strand break (SSB) accumulates in the failing heart and that SSB accumulation induces cell-autonomous inflammation through activation of DNA damage response (DDR) signaling pathway. Using alkaline- and neutral comet assay, we found that SSB is increased in the failing heart of pressure overload. Using in vitro model, we found that SSB accumulation activates ataxia talengiectasia mutated (ATM) kinase, which in turn induces nuclear translocation of NF-kB and increases the expression of inflammatory cytokines. Our findings suggest that SSB accumulation in cardiomyocytes plays an important role in the pathogenesis of heart failure by activating DDR pathway and subsequent cell-autonomous inflammation. SSB accumulation is supposed to be characteristic to post-mitotic cells like cardiomyocytes because unrepaired SSB usually develops into DNA double strand break and lead to catastrophic cellular death in mitotic cells. Approaches targeting efficient SSB repair or DDR pathway may become a novel therapeutic strategy against heart failure.
Abstract 56: Candesartan And Valsartan, Contrary To Irbesartan, Are Potent Biased Antagonists Of Adrenal (beta)arrestin-dependent, Angiotensin II Type 1 Receptor-induced Aldosterone Production And Improve Cardiac Function Post-myocardial Infarction
Authors: Lymperopoulos Anastasios , Walklett Karlee , Dabul Samalia , Siryk Ashley , Sturchler Emmanuel , McDonald Patricia , Rengo Giuseppe , Koch Walter J
Published: July 2021

Introduction:
The scaffolding protein βarrestin1 (βarr1) by the angiotensin II (AngII) type 1 receptor (AT
1
R) mediates AngII-induced aldosterone production in vitro and physiologically in vivo, thereby exacerbating heart failure (HF) progression post-myocardial infarction (MI). Herein, we sought to investigate the relative potency of various AT
1
R antagonist drugs (sartans) at inhibiting βarr vs. G protein activation and hence aldosterone production in vitro and in vivo. We also investigated the alterations in plasma aldosterone levels conferred by these agents and their impact on cardiac function of post-MI rats.

Methods:
For the in vitro tests, transfected CHO and adrenocortical H295R cells were used. For in vivo studies, post-MI rats overexpressing βarr1 in their adrenals received 7-day-long treatments with the drugs of interest.

Results:
Among the sartans tested, candesartan and valsartan were the most potent βarr activation and βarr-mediated aldosterone production inhibitors in vitro, as well as the most “biased” antagonists towards βarr vs. G-protein inhibition. Conversely, losartan and irbesartan were the least potent βarr inhibitors and the least “biased” antagonists towards βarr inhibition. These in vitro findings were corroborated in vivo, since candesartan and valsartan, contrary to irbesartan, caused significant plasma aldosterone reductions in post-MI rats. Accordingly, cardiac ejection fraction (EF) and contractility were significantly augmented in candesartan- and valsartan-treated rats (EF: 41.1±1% and 40±1% respectively, vs. 35±0.3% for saline-treated), but further deteriorated in irbesartan-treated post-MI rats (EF: 32±1%, n=7 rats/group).

Conclusions:
These findings provide important insights that might aid pharmacotherapeutic decisions (i.e. individual agent selections) involving this commonly prescribed cardiovascular drug class (sartans).
Abstract 61: miR-28 Induces Oxidative Stress via Nrf2 in Right Ventricular Failure but not in Left Ventricular Failure
Authors: Reddy Sushma , Hu Dong-Qing , Zhao Mingming , Blay Eddie , Fajardo Giovanni , Bernstein Daniel
Published: July 2021

MicroRNAs (miRs) are crucial regulators of cardiac remodeling in left ventricular hypertrophy and failure (LVH/LVF). However, there is minimal data on their role in right ventricular hypertrophy and failure (RVH/RVF), a risk for patients with congenital heart disease or pulmonary hypertension. Utilizing a murine model of RVH/RVF, we have described RV-specific overexpression of miR-28, not found in LVH/LVF. We used this model to evaluate miR-28 regulation of its downstream target Nrf2, a master regulator of antioxidant defenses, and a potential mechanism of the enhanced susceptibility of the RV to fail under pressure overload.

Methods:
miR-28 and Nrf2 gene and protein expression and ROS production and antioxidant defenses were assessed at 10d in RVH/RVF (pulmonary artery banding) and LVH/LVF (aortic banding). miR-28 was overexpressed in HEK293 cells and Nrf2 and ROS production assessed. Plasma miRs were also profiled.

Results:
Mice developed RVH by d4, at which time miR-28 was not increased vs. sham, and RVF by d10, when miR-28 was increased 2-fold. This was accompanied by decreases in Nrf2 gene (2-fold) and protein (0.4±0.2 vs. 0.8±0.1, p<0.05) expression, Nrf2-regulated SOD expression (2-fold), and SOD activity (80±15% vs. 90±18%, p<0.05). ROS production (4HNE) was increased (1.5±0.1 vs. 1.0±0.1, p<0.05). In contrast, at the same stage of LVH/LVF, miR-28 is not increased, Nrf2 expression is increased (0.45±0.2 vs. 0.1±0.02, p<0.05) and SOD is unchanged. Lentiviral miR-28 overexpression in HEK293 cells showed downregulation of Nrf2, SOD and heme oxygenase expression (1.6-2.1 fold) with a 35% increase in ROS production (p<0.05). Finally, plasma miR-28 decreased with the progression from RVH to RVF in mice, and this was confirmed in children undergoing pulmonary valve replacement for RV failure (patients vs controls, n=4/group).

Conclusions:
Our data show that RV-specific miR-28 enhances RVH/RVF through suppression of Nrf2 signaling and increased oxidative stress. Although we did not find Nrf2 downregulation at the same stage of LVH/LVF, others have shown this at 4-6 wks, suggesting this process may occur earlier in RVF vs. LVF. Finally, miR-28 plasma expression may be a biomarker for early RVF in patients.
Abstract 62: The Coxsackie Virus B3 modulates Cardiac Ion Channels
Authors: Seebohm Guiscard , Steinke Katja , Henrion Ulrike , Ettischer Nicole , Sachse Frank , Rohbeck Matthias , Klingel Karin , Strutz-Seebohm Nathalie
Published: July 2021

Infections with coxsackieviruses of type B (CVB) induce severe forms of acute and chronic myocarditis that are often accompanied by ventricular arrhythmias. The mechanisms underlying the development of virus-induced, life-threatening arrhythmia, remain largely elusive. Here, we show time-dependent CVB3-induced modulation of the cardiac ion channels Kv7.1, hERG1 and CaV1.2 in vitro. Channel protein localizations within cells and plasma membrane abundance are altered in infected mouse cardiac cells. In silico analyses of infected human myocytes suggest increased risk of arrhythmogenesis. These modifications are attenuated by the common Asian polymorphism KCNQ1-P448R, a genetic determinant preventing coxsackievirus-induced effects in vitro. This study provides a previously unknown explanation for the development of arrhythmias in enteroviral myocarditis, which will help to develop therapeutic strategies for arrhythmia treatment.
Abstract 63: Potential Genetic and Pharmacological Protection from Virus-induced Ventricular Arrhythmias and Sudden Cardiac Death
Authors: Strutz-eebohm Nathalie , Steinke Katja , Henrion Ulrike , Rohbeck Matthias , Klingel Karin , Seebohm Guiscard
Published: July 2021

In patients as well as in mouse models, enteroviral infections, especially Coxsackie group B viruses (CVB1-6), frequently induce ventricular arrhythmias and sudden cardiac death. The cardiac action potential requires proper function of cardiac ion channels. CVB3 alters Kv7.1 channel trafficking potentially leading to changes in action potentials and increasing likelihood of arrhythmias. Genetic variants of cardiac ion channels can cause changes in channel trafficking that may preserve from CVB3 modulations and present an evolutionary advantage. Here, we show that a common polymorphic Kv7.1 channel variant uses alternative trafficking pathways and may thus exert a benefit during CVB3 infections. Genetic and pharmacological disruption of a CVB3-stimulated Serum- and Glucocorticoid inducible Kinase 1 (SGK1) pathways blunts Kv7.1 channel dysfunctions.

Our results suggest that escape from CVB3-induced SGK1-stimulation by genetic variation in Kv7.1 may be protective and inhibition of SGK1 may present a pharmacological approach to reduce the pro-arrhythmic risk associated with acute coxsackievirus infections.
Abstract 72: Pka Is A Master Regulator Of Pathological Cardiac Hypertrophy
Authors: Zhang Xiaoying , LI Ying , Tang Mingxin , Ai Xiaojie , Szeto Christopher , Hua Xiang , Chen Xiongwen
Published: July 2021

Aims:
The role of PKA in pathological cardiac hypertrophy (PCH) is not clear. The literature suggests both prohypertrophic and antihypertrophic effects of PKA. Furthermore, there are endogenous PKA inhibitors, PKI, highly expressed in the heart to regulate PKA activity but their roles in PCH have not been studied. We aim to explore the role of PKI/PKA in PCH induced by isoproterenol, phenylephrine, angiotensin II and pressure overload.

Methods and Results:
1. PKIα and PKIγ were highly expressed in the heart but only PKIα was reduced by transaortic banding (TAB); TAB induced a significant increase in cardiac PKA activity at 1 week post TAB. 2. Four transgenic mouse lines with high (HE), medium (ME), low (LE) and very low (VLE) expression of PKI-GFP were obtained with the inhibition of maximum PKA activity induced by 1μM cAMP by 95%, 57%, 20% and 10% in the cardiac homogenates; 3. In the VLE hearts, some myocytes were PKI-GFP+ and some were PKI-GFP-, GFP- LVMs had significantly larger surface area than GFP+ LVMs; 4. PKA inhibition by PKI-GFP abolished PCH induced by isoproterenol, phenylephrine, angiotensin II in HE mice; 5. TAB for 8 weeks did not change HW/BW, myocyte cross-sectional area and myocardial fibrosis in HE mice but induced significant increases in HW/BW, myocyte cross-sectional area, myocardial fibrosis and depressed cardiac fractional shortening in control mice. 6. In cultured neonatal rat ventricular myocytes, PKI-GFP prevented myocyte hypertrophy induced by isoproterenol (ISO), phenylephrine (PE) and angiotensin II, as evidenced by no significant increases in protein synthesis (protein/DNA ratio), myocyte surface area, sarcomere organization. 7. PKI-GFP in NRVMs prevented the translocation of NFAT3 and HDAC5 induced by ISO and PE and increased the secretion of antihypertrophic ANF at baseline; 8. TAB induced PKA-dependent phosphorylation of GSK-3α and GSK-3β, inactivating them to relieve their antihypertrophic effect and promote protein synthesis (increased phosphorylation of mTORC1, eIF-4EBP1, p70 S6K); PKA inhibition abolished these effects.

Conclusions:
PKA is regulated by PKI and is a master regulator of PCH induced by pressure overload.
Abstract 78: Late Gadolinium Enhancement on Cardiac MRI Identifies Ventricular Dysfunction and Regional Myocardial Dyssynchrony in Patients with Univentricular Heart Diseases
Authors: Okuyama Michihiro , Ishigami Shuta , Ousaka Daiki , Kobayashi Junko , Kasahara Shingo , Sano Shunji , Oh Hidemasa
Published: July 2021

Backgrounds:
The impact of myocardial fibrosis on cardiac performance and clinical outcomes in patients with a functional single ventricle before stage-3 operation is unknown.

Objective:
The purpose of this study is to investigate the prognostic value of myocardial fibrosis identified by cardiac magnetic resonance imaging (cMRI) in patients with univentricular heart diseases.

Methods:
Consecutive 23 patients undergoing staged-3 surgical palliation with single ventricle physiology were prospectively scheduled to have cMRI study with late gadolinium enhancement (LGE) imaging and ventricle circumferential strain were examined.

Results:
Of 23 patients (mean age 3.3±0.9 years), 6 (26%) had positive late gadolinium enhancement (LGE+) in the ventricular myocardium, median percent LGE was 3.0% (interquartile range 3.0% to 14.0%). Pre-Fontan examinations revealed that patients with LGE+ had increased end-diastolic volume index (142.8 ml/BSA vs. 113.8 ml/BSA; P=0.02), increased end-systolic volume index (101.0 ml/BSA vs. 72.2 ml/BSA); P=0.01) compared with those without LGE (LGE-). Patients with LGE have shown to have lower ventricular circumferential strain compared with the area without LGE (basal: −1.9±1.9% vs. −4.0±3.0%, P=0.046; mid: −3.9±2.1% vs −8.0±3.9%, P=0.007; apical: −3.9±2.4% vs. −8.2±2.8%, P=0.004). In contrast to LGE- group, patients in LGE+ group had decreased right ventricular ejection fraction (27.7±8.8% vs. 38.2±8.4%; P=0.02) as well as higher levels of BNP (99.2±75.7 pg/ml vs. 32.6±44.3 pg/ml, P=0.02). In addition, patients with LGE+ had higher score of Ross classification (2.5±0.55 vs. 2.0±0, P=0.02) and New York University Pediatric Heart Failure Index (11.0±3.5 vs. 7.8±1.1, P=0.01) than in LGE- group. Age at stage-2 palliation was significantly older in patients with LGE+ group than LGE- subjects (16.8±16 months vs. 8.8±3.4 months, P=0.03).

Conclusion:
In this pre-stage-3 cMRI study, the age to stage-2 palliation may attribute to substantial myocardial fibrosis. The area of LGE was associated with impaired regional circumferential strain as well as disturbed ventricular performance. This novel strategy may provide a possible prognostic value of latent myocardial dysfunction after staged palliation.
Abstract 8: Transplantation of Mouse Adipose Derived Stem Cell Sheet into Infarcted Rat Myocardium Increase Cell Engraftment and Cardiac Function
Authors: Kim Jong-Ho , Joo Hyung Joon , Seo Ha-Rim , Cui Long-Hui , Kim Mi-Na , Choi Seung-Cheol , Park Chi-Youn , Choi Ji-Hyun , Hong Soon Jun , Lee Jeong Ik , Lim Do-Sun
Published: July 2021

Background:
Cell sheet technology has magnified as an important transplantation skill. Mouse adipose derived stem cells (mADSCs) can secrete various growth factors, which promote the repair of damaged cardiomyocyte and protecting cells from death. In addition, autologous cell source to easily obtain from patients are promising candidates for cell therapy in cardiovascular field.

Methods:
mADSCs were confirmed stem cell properties and secreted cytokines were evaluated in vitro. Eighteen acute myocardial infarction (AMI) rats were divide into 3 group; sham (n=6), suspended mADSCs (n=6), and mADSCs sheet (n=6) groups. In the mADSCs sheet group, 60х106 cells were cultured for 2 days onto temperature-responsive polymers and the sheets were then transplanted over the infarct region. In additional, the sheet was made of carboxyfluorescein diacetate succinimidyl ester (CFDA) -labelled mADSCs to confirmed cell survival. Engraftment and differentiation were blindly assessed after 28 days.

Results:
The mADSCs expressed Sca-1+ and represented multi-differentiation potential. Interestingly, EGF and IGF levels significantly increased in the mADSCs sheet. Significant improvements in ejection fraction and fraction shortening value were observed in the mADSCs sheet and suspended mADSCs groups compared with the sham group at 14 and 28 days. But, it was not higher significance level in the mADSCs sheet group than in the suspended mADSCs group. Engraftment range and fibrosis area of infarct region were significantly higher in the mADSCs sheet group compared to the other two groups at 4, 14 and 28 days. In significant expressed cytokines (bFGF, IL-1a, IL-1ra, CT-1, EGF, TGFb1, IGF-1, IGF-2 and MCP-1) were observed in the mADSCs sheet group compared with the other 2 groups at 28 days after transplantation. In addition, in the mADSCs sheet was confirmed endothelial differentiation by Von Willebrand factor (vWF) at 4, 14 and 28 days.

Conclusions:
Transplantation of mADSCs sheet into rat infarcted myocardium increased engraftment and survival of transplanted cells. The mADSCs sheet is very useful for the study of stem cell proliferation and differentiation as well as for cell therapy in cardiovascular field.
Abstract 86: Cardiomyocyte-Specific Ablation of Nuclear Respiratory Factor 1 in the Mouse Leads to Dysregulation of Mitochondrial Biogenesis, Apoptosis, and Heart Failure
Authors: Keenan Jeffrey E , Sulliman Hagir , Ulrich Allison , Mao Lan , Piantadosi Claude A
Published: July 2021

The DNA-binding transcription factor Nuclear Respiratory Factor 1 (NRF1) regulates mitochondrial homeostasis. Its constitutive ablation in the mouse is embryonically lethal (~E3.5). This has limited our understanding of NRF1 functionality in the heart, where mitochondrial dysfunction is often a major pathogenic factor. Therefore, we generated conditional cardiomyocyte-specific NRF1 knockout mice (MYH6-mer-Cre-mer-NRF1fl/fl or NRFfl/fl) to elucidate the role of cardiac NRF1. Two weeks after NRF1 silencing, echocardiography of NRF1fl/fl hearts revealed significant reductions in left ventricular fractional shortening (Figure A). Histology demonstrated degradation of cellular structural integrity and nuclear condensation (Figure B), with a high number of TUNEL positive nuclei compared to littermate controls (MYH6-mer-Cre-mer-NRF-1wt), indicative of apoptosis (37.8% vs. 1.1%, p < 0.001). The mRNA and protein levels of key mediators of mitochondrial biogenesis were evaluated by real-time RT-PCR and immunoblotting (Figure C & D). Compared to littermate controls, there was down-regulation of the mitochondrial encoded NADH dehydrogenase 1, implying a reduction of functional mitochondrial mass. Key biogenesis regulators PGC1-α (protein only), Nfe2l2, and NRF2 were also reduced. In total, these data support that dysregulation of mitochondrial biogenesis after loss of NRF1 results in cardiomyocyte apoptosis and reduced left ventricular function. These findings and further delineation of the mechanisms involved should lay the foundation for the exploitation of NRF1 as a therapeutic target in heart failure.
Abstract 90: Bendavia (MTP-131), a Novel Mitochondria-Targeting Peptide, Improves ADP-Stimulated Mitochondrial Respiration in Cardiomyocytes Isolated From Dogs with Chronic Heart failure
Authors: Sabbah Hani , Gupta Ramesh C , Rastogi Sharad , Mohyi Paula , Szekely Kristina
Published: July 2021

Background:
Mitochondria (MITO) of failed human hearts and hearts of dogs with experimental heart failure (HF) manifest structural and functional abnormalities characterized by hyperplasia and reduced organelle size and reduced respiration. These abnormalities lead to reduced ATP synthesis that adversely impacts LV function. We previously showed that chronic therapy (3 months) with Bendavia (MTP-131), a novel mitochondria-targeting peptide, improves LV systolic function in dogs with heart failure (HF), reverses MITO abnormalities and normalizes mitochondria ATP synthesis in myocardium from Bendavia-treated HF dogs. In the present study we examined the direct effects of Bendavia on mitochondria ADP-stimulated state 3 respiration in freshly isolated cardiomyocytes from dogs with advanced chronic HF.

Methods:
Cardiomyocytes were isolated from LV free wall of 3 untreated dogs with HF produced by intracoronary microembolizations (LV ejection fraction <30%). A standard collagenase-based enzymatic process was used for isolation that yielded ~70% viable rod-shaped cardiomyocytes that excluded trypan blue. Equal aliquotes of cardiomyocytes were incubated in 0, 0.01, 0.10, 1.0 and 10 μM concentration of Bendavia for one hour at 37°C. At the end of incubation, ADP-stimulated state-3 respiration was measured using a Clark electrode system and quatified in nAtom Oxygen/min/mg protein.

Results:
State-3 respiration in the absence of Bendavia (Vehicle-Control) was 248±9 nAtom Oxygen/min/mg protein. Compared to vehicle-control, incubation of failing cardiomyocytes with Bendavia significantly increased state-3 respiration to 303±33 at 0.01 μM, p<0.05; 405±39 at 0.10 μM, p<0.05; 371±28 at 1.0 μM, p<0.05; and 346±29 at 10.0 μM, p<0.05.

Conclusions:
Results of this study indicate that the effects of Bendavia on mitochondrial respiration in cardiomyocytes is direct and not a consequence of improved global LV structure or function. Furthermore, the results indicate that the improvement in mitochondrial respiration after treatment with Bendavia can occur early after initiation of therapy (within one hour) and is dose-dependent up to concentrations of 0.10 μM.
Abstract 92: Sirtuin 2 mediated-deacetylation regulates cellular iron homeostasis
Authors: Yang Xiaoyan , Vassilopoulos Athanassios , Park Seong-Hoon , Gius David , Ardehali Hossein
Published: July 2021

Background:
Sirtuins (SIRTs) are NAD+-dependent deacetylases, which regulate energy metabolism and response to oxidative stress in the heart. Iron is essential for these processes but is toxic when present in excess. However, whether SIRTs are involved in maintaining cellular iron homeostasis is not known. SIRT2 is among the least characterized SIRTs and is mainly present in the cytoplasm. We hypothesized that SIRT2 is required for cellular iron homeostasis.

Methods and Results:
Iron content was significantly lower in SIRT2-/- mouse embryonic fibroblasts (MEFs) compared to SIRT2+/+ MEFs (non-heme iron: 0.073 vs. 0.060 nmol/μg protein, p=0.02), andlevels of ferroportin-1 (FPN1), the major cellular iron exporter, was significantly increased in SIRT2-/- MEFs. Similarly, silencing SIRT2 in HepG2 cells decreased cellular iron levels and increased FPN1 expression, indicating that enhanced FPN1 with SIRT2 downregulation drove iron export and caused a reduction in cellular iron levels. Furthermore, iron export assays showed that iron export was increased in HepG2 cells with SIRT2 knockdown. To investigate the underlying mechanism, we focused our studies on nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a known regulator of FPN1. Our results demonstrated that Nrf2 is upregulated and translocates into the nucleus in SIRT2-/- MEFs and knocking down Nrf2 in SIRT2-/- MEFs reverses iron deficiency. Furthermore, Nrf2 is acetylated by P300/CBP and can be deacetylated by SIRT2. Finally, to confirm the role of SIRT2 in iron regulation, cellular heme and non-heme iron in the heart (major iron-consuming organ) and liver (major iron-storage organ) were measured in wild type (WT) and SIRT2-/- mice. Heme and non-heme iron content were significantly decreased in SIRT2-/- mouse livers compared to WT livers (heme: 2.25 vs. 1.65 nmol/mg protein, p=0.002; non-heme iron: 0.073 vs. 0.064 nmol/μg protein, p=0.03). Furthermore, heme levels were also significant decreased in the heart, while non-heme iron was not significantly altered.

Conclusions:
Our results suggest that SIRT2 regulates cellular iron homeostasis by deacetylating NRF2 and altering iron export through FPN1.
Abstract 98: Reducing Mitochondrial, But Not Cytosolic Iron, Protects The Heart Against Ischemia-reperfusion Injury
Authors: Chang Hsiang-Chun J , Wu Rongxue , Ardehali Hossein
Published: July 2021

Introduction:
Iron is essential for the activity of a large number of cellular proteins, but excess free iron can cause cellular damage through production of reactive oxygen species (ROS). Mitochondria are the major site of cellular iron homeostasis, and we recently showed the mitochondrial iron export is mediated by ATP-binding cassette protein-B8 (ABCB8). The role of mitochondrial iron in ischemia-reperfusion (I/R) injury in the heart has not been examined. We hypothesize that mitochondrial iron has a critical role in I/R damage and a reduction of mitochondrial iron is protective against I/R injury through a reduction in ROS.

Results:
Cardiomyocyte-specific ABCB8 transgenic (TG) mice had significantly lower mitochondrial iron in the heart than nontransgenic (NTG) littermates at baseline, but their cardiac function and the expression of key antioxidant systems were indistinguishable from NTG littermates. To study the role of mitochondrial iron in I/R injury, we subjected ABCB8 TG mice to I/R. TG mice displayed significantly less apoptosis compared to NTG littermates (11.76% vs. 17.63%, p<0.05, n=4-6) and had significantly reduced lipid peroxidation products 48 hours after I/R.

To further confirm that our in vivo finding was due to reduced mitochondrial iron, we studied the effect of pharmacological reduction of mitochondrial iron in vitro. 2,2-bipyridyl (BPD) is a mitochondria-accessible iron chelator while deferoxamine (DFO) has poor penetrance into mitochondria. Treating rat cardiomyoblasts H9C2 with BPD but not DFO significantly reduced chelatable mitochondrial iron, as measured by staining cells with rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzyl ester. In addition, BPD but not DFO pretreatment protected cells against H2O2 induced cell death (p<0.05). BPD treatment in mice decreased baseline mitochondrial iron and significantly preserved cardiac function after I/R.

Conclusions:
Our findings demonstrate that selective reduction in mitochondrial iron is protective in I/R injury, and show that mitochondrial iron is a source of ROS and cellular damage in I/R. Thus, targeting mitochondrial iron with selective iron chelators, as studied in our system, may provide a novel approach for treatment of ischemic heart disease.
Abstract O-2: The Role of Post-translational Modifications in SERCA2a-related Cardiac Dysfunction
Authors: Kho Changwon , Jeong Dongtak , Lee Ahyoung , Mitsuyama Shinichi , Oh Jae Gyun , Hajjar Roger
Published: July 2021

The cardiac sarcoplasmic reticulum calcium ATPase (SERCA2a) has become a validated target for the treatment of heart failure (HF). The relationship between reduced SERCA2a activity and decreases in protein expression in the setting of HF has been found to be non-linear and the toxic intracellular milieu in HF contributes to SERCA2a’s dysfunction. Post-translational modification (PTM) of SERCA2a has been recently described to as an important mechanism that can explain a reduction in SERCA2a activity in HF. Based on a comprehensive proteomic analysis, we found that the levels and activity of SERCA2a in cardiomyocytes are modulated in parallel with the levels of small ubiquitin-like modifier type 1 (SUMO-1). Moreover, our work has shown that SUMO-1 plays a critical role in protecting SERCA2a from pathological conditions (Kho et al, Nature, 2011). More recently, we demonstrated that SUMO-1 gene transfer and its combination with SERCA2a led to a reversal of HF in a porcine model of ischemic induced HF (Tilemann et al, Sci Transl Med, 2013). In our analysis of SERCA2a PTM in animal models of HF, we observed that SERCA2a is acetylated, and that this acetylation is more prominent in failing hearts. The acetylation of SERCA2a was validated by acetylation assays with acetyltransferase and HDAC inhibitors. We identified several lysine residues on SERCA2a for susceptible to acetylation. In addition, we found that Sirt1 enzyme deacetylates SERCA2a. Sirt1 down-regulation in HL-1 cells using small interfering RNA increased SERCA2a acetylation and thereby decreased its activity. Moreover, SERCA2a acetylation was increased when Sirt1 was depleted by recombinant adeno-associated virus carrying short hairpin RNA for Sirt1 in mice model, which reflected a decrease in intensity of interaction between Sirt1 and SERCA2a. Reduced acetylation was accompanied by an increase in SERCA2a SUMOylation in the heart. Decreased acetylation, combined with increased SUMOylation, of SERCA2a may contribute to the cardioprotective effects of Sirt1. Our results show that SERCA2a acetylation increases during HF and negatively impacts SERCA2a’s function, suggesting that the down-regulation of SERCA2a acetylation may afford a novel intervention in the setting of heart failure.
Abstract O-3: A β1-Adrenergic Receptor/β-Arrestin1-Regulatable MicroRNA, MiR-150 Protects the Mouse Heart from Ischemic Injury by Repressing Pro-apoptotic Egr2 and P2x7r
Authors: Kim Il-man , Tang Yaoping , Wang Yongchao , Park Kyoung-mi , Hu Qiuping
Published: July 2021

MicroRNA (miR)-150 is down-regulated in patients with acute myocardial infarction (AMI), atrial fibrillation, dilated and ischemic cardiomyopathy as well as in various mouse heart failure (HF) models. Circulating miR-150 has been recently proposed as a better biomarker of HF than clinically used markers such as brain natriuretic peptide. We recently showed that β-arrestin1-biased β1-adrenergic receptor (β1AR) cardioprotective signaling activated by the β-arrestin-biased β-blocker, carvedilol (Carv) stimulates the processing of miR-150 in the heart (see figure A). However, the potential role of miR-150 in ischemic injury and HF is unknown. Here, we show that genetic deletion of miR-150 in mice causes abnormalities in cardiac structural and functional remodeling after MI. The cardioprotective roles of miR-150 during ischemic injury were attributed to repression of the pro-apoptotic genes egr2 (zinc binding transcription factor induced by ischemia) and p2x7r (pro-inflammatory ATP receptor) [see figure B]. These findings reveal a pivotal role for miR-150 as a regulator of cardiomyocyte survival during cardiac injury. In conclusion, our study will help to stratify HF patients that may respond better to β-arrestin-biased β-blockers, which is guided by circulating levels of miR-150.
Abstract 101: MiR-4260 Promotes the Proliferation, Migration, and Tube Formation of Human Umbilical Vein Endothelial Cells
Authors: Sun Qi , Lv Dongcao , Zhou Qiulian , Bei Yihua , Xiao Junjie
Published: July 2021

MicroRNAs (miRNAs, miRs), endogenous small non-coding RNA, have been shown to act as essential regulators in angiogenesis which plays important roles in improving blood flow and cardiac function following myocardial infarction. The current study investigated the potential of miR-4260 in endothelial cell function and angiogenesis using human umbilical vein endothelial cells (HUVEC). Our data demonstrated that overexpression of miR-4260 was associated with increased proliferation and migration of HUVEC using EdU incorporation assay (17.25%±1.31 vs 25.78%±1.24 in nc-mimics vs miR-4260 mimics, respectively) and wound healing assay, respectively. While downregulation of miR-4260 inhibited the proliferation (17.90%±1.37 vs 10.66%±1.41 in nc-inhibitor vs miR-4260 inhibitor, respectively) and migration of HUVEC. Furthermore, we found that miR-4260 mimics increased (129.75±3.68 vs 147±3.13 in nc-mimics vs miR-4260 mimics, respectively), while miR-4260 inhibitor decreased the tube formation of HUVECs in vitro (123.25±2.17 vs 92±4.45 in nc-inhibitor vs miR-4260 inhibitor expression, respectively). Our data indicate that miR-4260 contributes to the proliferation, migration and tube formation of endothelial cells, and might be essential regulators for angiogenesis. Further study is needed to investigate the underlying mechanism that mediates the role of miR-4260 in angiogenesis by identifying its putative downstream target genes.
Abstract 123: Cardiac Beta-arrestin2 Promotes Contractility by Enhancing SERCA2a SUMOylation and Activity
Authors: Lymperopoulos Anastasios , Jafferjee Malika , Reyes Valero Thairy , Marrero Christine , McCrink Katie A , Brill Ava , Gao Erhe , Koch Walter J
Published: July 2021

Heart failure (HF) is the number one killer disease in the western world and new and innovative treatments are needed. Sarco(endo)plasmic reticulum Ca
2+
-ATPase (SERCA)-2a is a crucial, for contractile function, calcium-handling protein expressed in the mammalian myocardium and its downregulation is one of the molecular hallmarks of chronic HF. Its activation is part of the signaling mechanism by which the β
1
-adrenergic receptors (ARs) increase cardiac contractility. Agonist-bound β
1
ARs however, like most G protein-coupled receptors (GPCRs), undergo functional desensitization/internalization due to the actions of βarrestin1 or -2. These two arrestins are universal GPCR adapter proteins, mediating G protein-independent signaling via multi-protein scaffolding, and, among the cellular processes they can regulate, is protein SUMO (small ubiquitin-like modifier)-ylation, which generally increases protein stability/levels. In the heart, βarrestin1 appears detrimental, whereas βarrestin2 beneficial, for structure and function post-myocardial infarction (MI). Post-MI βarrestin1 knockout mice also display elevated SERCA2a activity and better contractility than post-MI wild type mice. In addition, reduced cardiac SERCA2a SUMOylation is known to underlie its downregulation in HF, decreasing cardiac contractility. Thus, in the present study, we sought to investigate a potential involvement of cardiac β
1
AR-activated βarrestins in regulation of SERCA2a SUMOylation and activity. By studying individual βarrestin knockout heart extracts, we found that βarrestin2, but not βarrestin1, interacts with SERCA2a in the mouse heart in vivo, promoting the latter`s SUMOylation and activity. This interaction is direct, as indicated by pull-down and FRET experiments. Finally, via in vitro studies in the cardiomyocyte-like cell line H9c2, we found that this interaction is both β
1
AR-, and beta-agonist-specific, and leads to increased Ubc9-dependent SERCA2a SUMOylation, which, in turn, acutely enhances SERCA2a activity in H9c2 cells. These results suggest that βarrestin2, presumed to also decrease cardiac function by desensitizing βARs, may actually (directly) enhance cardiac contractility, thereby opposing βarrestin1 in that regard.
Abstract 128: Network-based Approaches to Identify Novel Regulators of Heart Failure
Authors: Rau Christoph D , Romay Milagros C , Wang Jessica , Ren Shuxun , Wang Yibin , Lusis Aldons J
Published: July 2021

Heart failure is a highly heterogeneous disorder characterized by the interactions of multiple environmental and genetic factors. While reductionistic approaches have made significant inroads into characterizing the pathophysiology of the syndrome, they are unable to properly dissect the complex interactions between sets of genes and pathways which result in the emergent phenotypes . Systems genetics offers a means by which these interactions may be identified and explored. We have developed a resource, the Hybrid Mouse Diversity Panel (HMDP) to perform systems-level analyses in mice. Nine week old female mice from 93 unique inbred lines of the HMDP were give 30 ug/g/day of isoproterenol through an abdominally implanted Alzet micropump. After three weeks, mice were sacrificed along with age-matched controls. A portion of the left ventricle was arrayed on an Illumina Mouse Ref 8.0 platform.

Maximal Information Component Analysis was used to construct gene networks, and a module of 41 genes was identified which shows strong correlation to a number of important phenotypic traits, including heart weight and cardiac fibrosis. This module contains a number of genes of interest, including Lgals3, a diagnostic marker for heart failure. Through the use of structural equation modeling, we identified several key genes within the module for further analysis, the most important of which is the metalloprotease Adamts2.

We have performed a series of in vitro analyses demonstrating the important role of Adamts2 in this module using neonatal rat ventricular myocytes. Knockout of Adamts2 results in an amelioration of the hypertrophic response to catecholamine stimulation as well as a reduction of hypertrophic markers such as Nppa and Nppb. Furthermore, we observe that other genes in the module no longer respond to catecholamine stimulation after knockdown of Adamts2.
Abstract 15: Global RNA Splicing Regulation in Cardiac Maturation
Authors: Gao Chen , Ren Shuxun , Lee Jae-Hyung , Hsiao Yun-Hua Esther , Xiao Xinshu (Grace) , Chen Jau-nian , Nakano Atsushi , Wu Joseph C , Wang Yibin
Published: July 2021

Background:
The complexity of transcriptome and proteome is contributed by alternative splicing of mRNA. Altered mRNA splicing is implicated in both development and disease. However, the change of alternative mRNA splicing during cardiomyocytes maturation is unknown, and the regulatory mechanisms remain unexplored.

Methods and Results:
Using deep RNA-Sequencing, we identified global alternative splicing changes associated with both cardiac development and pathological remodeling in mouse heart. Further, we identified a highly conserved splicing regulator-RBFox1 to be significantly induced during zebrafish, mouse and human cardiac maturation. RBFox1 expression was also detected in cardiomyocytes derived from both mouse and human embryonic stem cells but at much lower levels comparing to adult heart. In zebrafish embryos, inactivation of RBFox1 caused cardiomyocyte maturation defects. Expression of RBFox1 in cultured neonatal cardiomyocytes was sufficient to promote maturation by reducing fetal marker gene expression while increasing calcium handling gene expression including RyR and promoting sarcomere organization. Deep RNA-Sequencing analysis showed that RBFox1 expression promoted alternative splicing in genes involved in calcium cycling, blood vessel development and muscle contraction. Finally, we identified a highly conserved mutually exclusive alternative splicing event of transcription factor MEF2 to be a direct downstream target of RBFox1. Expression of individual MEF2 splicing variants led to different cardiac developmental phenotypes in zebrafish, indicating their different transcriptional activities.

Conclusion:
Our study provided the first comprehensive analysis of mRNA splicing regulation in heart during post-natal development and heart failure, and identified RBFox1 as a key regulator for alternative RNA splicing during cardiomyocytes maturation. Further exploration of RBFox1 mediated RNA splicing regulation in heart may yield novel insight to the underlying mechanisms of cardiac maturation and new approach to improve cell based therapy for heart diseases.
Abstract 151: Cell-based Therapies for Heart Failure: Changes in Cytokine Expression From Mesenchymal Stem Cells and Induced Pluripotent Stem Cell Derived Cardiomyocytes
Authors: Pandey Amitabh C , Lancaster Jordan , Harris David , Goldman Steven , Juneman Elizabeth
Published: July 2021

Mesenchymal stem cells (MSCs) use paracrine signaling to modulate the cellular microenvironment via expression of cytokines, chemokines, and adhesion molecules to aid and promote endogenous repair. Induced pluripotent stem cell derived cardiomyocyte (iPSC-CMs) and mesenchymal stem cells (MSCs) together may play a synergistic role in changing the microenvironment milieu to allow for endogenous cellular repair through paracrine signaling. Cytokine expression is involved in the progression of heart failure (HF). Using a rat model of HF, cell based therapies with a fibroblast embedded patch only, iPSC-CM patch, and MSCs via tail vein (IV) or intracardiac injections (IC) to the left ventricle (LV) were administered, and RNA was subsequently isolated, and real-time polymerase chain reaction (PCR) was performed for analysis of gene expression.

Evaluation of gene expression revealed significant increases in the expression of connexin 43 with iPSC-CMs (p<0.05). Expression of MMP9 was decreased with MSCs alone (p<0.05) but with the use of the patch with both cell types, its levels were significantly increased (p<0.05). Myosin heavy chain was seen to increase significantly with increasing cell numbers in iPSC-CM therapy (p<0.05). Markers of angiogenesis including vascular endothelial growth factor, angiopoietin, and insulin like growth factor were significantly increased with iPSC-CM patch therapy (p<0.05). Up-regulation of angiogenic cytokines and cardio-protective cytokines may help in slowing progression of HF. Interestingly, we also observed an increase in some markers, which were associated with HF. In conclusion, both iPSC-CM patch and MSCs altered signaling in the setting of HF, perhaps leading to improvement of at the cellular level. An iPSC-CM-based patch and MSCs as an adjuvant therapy may be able to play a role in the setting of HF as cellular therapeutic approach.
Abstract 153: Using the Embryonic Heart as an Instructive Template for Cardiac Tissue Engineering
Authors: Batalov Ivan , Jallerat Quentin , Feinberg Adam W
Published: July 2021

The engineering of highly aligned cardiomyocytes into functional heart muscle remains a primary challenge in cardiac tissue engineering. Researchers have shown that micropatterned topography and chemistry as well as mechanical and electrical gradients are all effective at inducing some degree of alignment. However, which approach works best in terms of electromechanical function of the engineered cardiac muscle is still an active area of research. Because formation of new heart muscle in mammals primarily occurs during cardiogenesis, we asked whether the embryonic heart could be used as an instructive template for the design of more effective cardiac tissue engineering scaffolds. Specifically, we hypothesized that micropatterns of fibronectin based on fibronectin fibril size and architecture in embryonic myocardium could improve cardiomyocyte alignment relative to 20 μm wide, 20 μm spaced fibronectin lines, a control pattern used widely in the literature. To test this, we first imaged the fibronectin matrix in the ventricles of day-5 embryonic chick hearts and imaged this in 3D using a multiphoton microscope. This fibronectin structure was then converted into a photomask for photolithography and subsequent patterning of fibronectin onto cover slips using microcontact printing. Samples with the biomimetic patterns or control patterns were seeded with embryonic chick cardiomyocytes, cultured for 3 days and then stained and imaged to visualize the myofibrils. Image analysis to quantify alignment showed that the ability of the biomimetic pattern to induce cardiomyocyte alignment increased with cell density, suggesting that cell-cell interactions play an important role in the formation of aligned embryonic myocardium. Disruption of the cadherins junctions using blocking antibodies confirmed this conclusion. In the future we will use human induced pluripotent stem cell-derived cardiomyocytes to engineer more clinically-relevant human heart muscle and analyze electromechanical function of the tissues including contractile force and action potential propagation.