bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 327: Utrophin Loss in the Absence of Dystrophin Induces Alterations in Cardiac-Related Protein Expression and Activity in the Heart and Muscle of Dystrophic Mice
Authors: Schneider Joel S , Ramachandran Jayalakshmi , Shanmugam Mayilvahanan , Crassous Pierre-Antoine , Gonzalez James P , Gordan Richard , Xie Lai-Hua , Beuve Annie V , Babu Gopal J , Peluffo Ruben D , Fraidenraich Diego
Published: July 2021

Duchenne Muscular Dystrophy (DMD) is an x-linked recessive disorder that affects 1:3500 males. The disease is characterized by the absence of the dystrophin protein, leading to progressive muscular degeneration in both skeletal and cardiac muscle. DMD is recapitulated in mouse models, the most severe model lacking both dystrophin and its compensatory homolog, utrophin (mdx:utro). This model mimics the onset of DMD much more rapidly than mdx. Nitric oxide (NO) synthesis is significantly diminished in DMD and mdx skeletal muscle. We have identified previously unreported functional differences in neuronal nitric oxide synthase (NOS1)-related nitric oxide production in cardiac musculature in mdx:utro mice, caused by the absence of utrophin. We have quantified mechanistic differences in L-Arginine mediated nitric oxide production via NOS1 in mdx and mdx:utro myocytes isolated via Langendorff perfusion. Interestingly, L-arginine transport levels were increased in the presence of this amino acid and occurred concurrently with increases in the number of mRNA transcripts for the low-affinity cationic amino acid transporter, CAT-2A. As CAT activity is an obligate process for L-Arginine entry to the cardiac myocytes, this may represent a potential compensatory mechanism related to decreased nitric oxide production.

We have also found manifestations of ectopic cardiac-related protein expression in the quadriceps muscle of mdx:utro mice. In these tissues, we have found ectopic expression of the cardiac isoform of the SERCA2a calcium pump predominantly expressed in the heart. Additionally, we have found expression of slow-twitch Type 1 cardiac myosin heavy chain, indicative of a fiber type switch event. We have shown that expression of both of these proteins is co-localized to specific muscle fibers. Our analyses indicate that the appearance of these proteins is directly correlated with the lowered expression of the fast-twitch calcium pump (SERCA1) and muscle fiber types (Type 2a and Type 2b).

These findings illustrate important molecular consequences of the absence of utrophin in mouse models of DMD. Identification of compensatory corrective targets may bear important therapeutic potential in muscular dystrophy and its associated cardiomyopathy.
Abstract 330: AT1R Conformation Mediates Mechanical Stress-Induced β-Arrestin Signaling
Authors: Tang Wei , Rockman Howard A
Published: July 2021

Wei Tang1, Howard A. Rockman1,2,3 1Departments of Medicine, 2Departments of Cell Biology, 3Departments of Molecular Genetics and Microbiology, Duke University Medical Center, DUMC 3104, 226 CARL Building, Durham, NC 27710, USA.

Introduction:
Recent work has shown that mechanical stretch induces β-arrestin-biased Angiotensin II Type 1 Receptor (AT1R) prosurvival cellular signaling. However, the precise molecular details for this are not fully understood. We set out to explore whether the conformation of the AT1R determines stretch-induced cellular signaling and whether this mechanism of stretch-mediated beta-arrestin signaling activates cell cycle progression.

Methods:
We used HEK 293 cells stably expressing AT1Rs with and without treatment with the nonionic triblock membrane sealant poloxamer 188 (P188). P188 is known to integrate into biological membranes and affect the local microviscosity of the membrane. Mechanical stretch was induced by hypotonic osmotic stress. β-arrestin signaling was examined by the level of phosphorylated ERK (pERK) with and without siRNA targeted to β-arrestin1/2, and cell cycle progression by the level of phosphorylated retinoblastoma (Rb).

Results:
Both Angiotensin II (AngII) and osmotic stress increased pERK. The increase in pERK by osmotic stress was abrogated in the presence of β-arrestin1/2 siRNA. P188 had no effect on AngII induced pERK, but completely blocked the increase in pERK with osmotic stress. Both AngII and osmotic stress caused the rapid increase phosphorylated Rb at the Ser780 site, which is known to be phosphorylated by cyclin-dependent protein kinase 4.

Conclusion:
Altering membrane viscosity blocks stretch-induced AT1R signaling indicating a possible role for receptor conformation in stretch-mediated β-arrestin signaling and cell cycle progression. Ongoing studies will directly measure receptor affinity states under ligand-activated vs. mechanical stretch-activated conditions.
Abstract 332: Nanostructure-Templated Alignment of Cardiomyocytes for 3-Dimensional Cardiac Tissue Engineering
Authors: Sleep Eduard , Mantei Jason , McClendon Mark , Hoxha Eneda , Kishore Raj , Stupp Samuel I
Published: July 2021

Introduction:
Heart muscle shows a significant amount of alignment and strategies for cardiac tissue engineering should aim at mimicking such topology. So far, conditions to align cardiomyocytes in three-dimensional (3D) engineered cardiac constructs include static and active mechanical stress and the application of a directional electric field. However, the question of how topological cues from the matrix in which cardiomyocytes are embedded affect their alignment has not been addressed yet. Here, we investigated how cardiomyocytes align and mature in novel 3D gels made out of peptide amphiphiles (PAs) that can be aligned at the nanostructure level.

Methods:
We seeded HL-1 cardiomyocytes, mouse ES cell-derived cardiomyocytes and human iPS cell-derived cardiomyocytes into PA solutions that were either or not aligned upon gellation. We assessed the alignment of the cardiomyocytes along with their maturation status by observing their structural proteins and the formation of functional syncitiums by connexin 43 expression and the propagation of calcium fluxes. We also tested whether the stiffness of the gel affected the above-mentioned parameters by changing the chemical structure of the PAs.

Results:
We found that cardiomyocytes aligned along the direction of the alignment of the nanostructures in the gel and that the alignment of the matrix contributed to the functional maturation of the construct. Moreover, we observed a relationship between the stiffness of the gels and the alignment of the cardiomyocytes.

Conclusions:
This study shows for the first time that the nanostructural features of 3D scaffolds can be exploited to create aligned cardiac constructs.
Abstract 339: Oxygen Blunts A2aR to Augment Lung Injury
Authors: Aggarwal Neil , King Landon , Polotsky Vsevelod
Published: July 2021

Introduction:
Mechanisms leading to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remain incompletely understood, yet the severity of illness and societal disease burden necessitate continued investigation. On-going lung inflammation and injury may occur due to inhibition of anti-inflammatory pathways in alveolar macrophages. With tissue injury, released adenosine preferentially activates the adenosine 2A receptor (A2aR), a pathway which has been implicated in mediating anti-inflammatory effects and tissue protection; however, modifiers of this pathway in macrophages are not well-described. In a murine model, we have observed that moderate levels of oxygen after intratracheal LPS increase and sustain macrophage inflammation. We hypothesize that moderate oxygen inhibits the A2aR pathway in macrophages to augment inflammation and lung injury.

Methods:
Murine model consists of intratracheal (IT) delivery of LPS, followed 12 hours later by 60% oxygen or 21% oxygen (RA) via sealed chamber.

Results:
Wild-type mice exposed to IT LPS (0.375 [[Unsupported Character - Symbol Font ]]g/g mouse) followed by 60% oxygen had significantly more lung injury at days 2-4 in comparison to mice exposed to LPS or 60% oxygen alone, with increased alveolar cell influx, histologic injury, and epithelial barrier permeability. 60% oxygen after LPS significantly reduced lung A2aR mRNA and alveolar cell intracellular cyclic AMP (IC cAMP), located directly downstream of A2aR. Reduction in IC cAMP also occurred in isolated macrophages exposed to both LPS and oxygen when compared to LPS alone; this reduction was reversed using a specific A2aR agonist, CGS 21680. Adding oxygen or
ZM241385
(A2aR antagonist) to LPS-exposed macrophages increased secretion of pro-inflammatory cytokines TNF-[[Unsupported Character - Symbol Font ]] and MIP-2, and expression of co-stimulatory molecules CD86 and CD40. Murine delivery of CGS 21680 (125ug/dose/mouse) every 24 hours starting concurrently with oxygen exposure reduced lung injury in LPS and oxygen-exposed mice.

Conclusion:
After LPS exposure, 60% oxygen acts to decrease activation of the A2aR pathway in macrophages which may directly exacerbate lung injury.
Abstract 35: Elevated MicroRNA-155 Targets HBP1 to Promote the Formation of Macrophage-Derived Foam Cell and This Can Be Effectively Suppressed by YY1/HDACs Complex
Authors: Jing Qing , Tian Fu-Ju , Li Qing , Zou Jun
Published: July 2021

Aims:
We aim to investigate the role of microRNAs in the formation of macrophage-derived foam cell.

Methods and results:
Using quantitative RT-PCR, we found that the level of microRNA-155 (miR-155), a macrophage-associated microRNA, was significantly increased both in plasma of atherosclerotic ApoE-/- mice and in macrophages isolated from this animal model. Indeed, oxLDL effectively induced expression and release of miR-155 in macrophages. We further identified that miR-155 was required to mediate oxLDL-induced lipid uptake and ROS production of macrophages. Importantly, ectopic overexpression and knockdown experiments identified that HMG box-containing protein 1(HBP1) is a novel target of miR-155. Knockdown of HBP1considerably enhanced lipid uptake and ROS production in oxLDL-induced macrophages, and overexpression of HBP1 effectively repressed these effects caused by miR-155 overexpression. Interestingly, bioinformatics analysis identified three YY1 binding sites in promoter region of pri-miR- 155. EMSA and ChIP analyses verified YY1 directly binding to its promoter region. Furthermore, quantitative RT-PCR analysis showed that YY1/HDAC2/4 complex negatively regulated the expression of miR-155 to suppress foam cell formation.

Conclusions:
In oxLDL-induced macrophages, elevated miR-155 directly targets HBP1 to promote lipid uptake and ROS production, and YY1/HDAC2/4 complex effectively inhibits foam cell formation mediated by miR-155. This study reveals a novel molecular mechanism in the atherosclerosis and suggests miR-155 is a potential therapeutic target.
Abstract 353: Sunitinib Induces Endoplasmic Reticulum Stress in Cardiomyocytes
Authors: Rees Meredith L , Taegtmeyer Heinrich
Published: July 2021

Introduction:
Chemotherapy-induced cardiac toxicities are the main obstacle for effective treatment of cancer, particularly in patients with pre-existing heart disease. This often results in premature termination of cancer treatment. Prominent among these agents is sunitinib (Sutent), which causes cardiac dysfunction in nearly 20% of cancer patients. It has recently been suggested that cardiac microvascular dysfunction, particularly loss of pericyte coverage, underlies sunitinib-induced cardiac dysfunction. However the exact mechanisms of sunitinib-induced cardiotoxicites are still unknown.

Results:
Neonatal rat ventricular myocytes were treated with increasing concentrations (100nM-5uM) of sunitinib. Irrespective of the dose, there was a significant increase in markers of ER stress (Grp78, Grp 94, Erp72 and phospo/total-eIF2a) after 24 hours of treatment. The ER stress inducer thapsigargin was used as a positive control. Concordant with an increase in ER stress was the activation of mTOR in sunitinib (but not thapsigargin) treated NRVMs, suggesting a possible role of mTOR-mediated proteotoxicity in sunitinib-induced cardiac dysfunction. Protein synthesis, evaluated by
14
C-phenylalanine incorporation into proteins, was moderately elevated in cells treated with sunitinib.

Conclusions:
In addition to impairment of angiogenesis and pericyte loss, sunitinib induces mTOR activation and ER stress. We propose that the cardiac myocyte secretes paracrine factors that stabilize the cardiac microcirculation. This process is disrupted by sunitinib and ultimately leads to heart failure.
Abstract 354: Prostaglandin D
2
-F Type Prostanoid Receptor Signaling Is a Novel Cardiomyocyte Survival Pathway
Authors: Katsumata Yoshinori , Sano Motoaki , Matsuhashi Tomohiro , Anzai Atsushi , Yan Cardex , Ito Kentaro , Shinmura Ken
Published: July 2021

Background:
Lipocalin-type prostaglandin D synthase (L-PGDS) is abundantly expressed on cardiomyocytes. We recently demonstrated that dexamethasone stimulates PGD
2
-dominated activation of prostanoid biosynthesis, thereby protecting hearts against ischemia/reperfusion injury. Here, we examined the downstream signaling responsible for cardioprotection mediated through PGD
2
-dominated activation.

Methods and Results:
(1) In cultured neonatal rat cardiomyocytes, PGD
2
strongly activates ERK in a dose-dependent manner, although canonical PGD
2
receptors, including DP (PGD
2
receptor) and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells) receptors, are hardly expressed on cardiomyocytes. (2) Interestingly, PGD
2
bounds to FP receptor (the canonical PGF
2
α receptor) with an affinity comparable to that for the DP receptor, and the FP receptor is abundantly expressed on cardiomyocytes. (3) PGD
2
-induced ERK activation is completely blocked by FP antagonist or siRNA-mediated knockdown of the FP, but not by DP and CRTH2 antagonist and siRNA-mediated knockdown of DP and CRTH2. (4) PGD
2
activates ERK in Langendorff perfused DP-knock out (KO) and CRTH2-KO mice hearts to comparable levels as those observed for wild-type hearts, while cannot activate it in FP-KO hearts. (5) Consistently, the cardioprotective effect of PGD
2
-dominated activation by dexamethasone was blunted in FP KO hearts. (6) Furtermore, genomewide gene expression profiles by microarray analysis and quantitative real-time RT-PCR analysis identified that Nrf-2 was the downstream target of L-PGDS-mediated PGD
2
biosynthesis. (7) In cultured cardiomyocytes, FP agonist stimulated Nrf2 nuclear translocation and consequently induced Nrf2-target genes expression in an ERK-dependent manner. (8) Finally, The cardioprotective effect by dexamethasone was completely abolished in Nrf-2 KO hearts.

Conclusion:
FP serves as a functionally relevant PGD
2
receptor in the hearts and PGD
2
-FP signaling plays a substantial role in the improvement of functional recovery after ischemia-reperfusion injury in the heart. Nrf-2 is a major effector molecule responsible for the cardioprotecton elicited by L-PGDS-derived PGD
2
.
Abstract 356: Krueppel-Like Factor 15 Regulates Wnt/β-Catenin Transcription and Controls Cardiac Progenitor Cell Fate in the Postnatal Heart
Authors: Noack Claudia , Zafiriou Maria P , Renger Anke , Schaeffer Hans J , Bergmann Martin W , Zelaráyan Laura C
Published: July 2021

Wnt/β-catenin signaling controls adult heart remodeling partly by regulating cardiac progenitor cell (CPC) differentiation. We now identified and characterized a novel cardiac interaction of the transcription factor Krueppel-like factor 15 (KLF15) with the Wnt/β-catenin signaling on adult CPCs.

In vitro mutation, reporter gene assays and co-localization studies revealed that KLF15 requires two distinct domains for nuclear localization and for repression of β-catenin-mediated transcription. KLF15 had no effect on β-catenin stability or cellular localization, but interacted with its co-factor TCF4, which is required for activation of β-catenin target gene expression. Moreover, increased TCF4 ubiquitination was induced by KLF15. In line with this finding we found KLF15 to interact with the Nemo-like kinase, which was shown to phosphorylate and target TCF4 for degradation.

In vivo analyses of adult Klf15 functional knock-out (KO) vs. wild-type (WT) mice showed a cardiac β-catenin-mediated transcriptional activation and reduced TCF4 degradation along with cardiac dysfunction assessed by echocardiography (n=10). FACS analysis of the CPC enriched-population of KO vs. WT mice revealed a significant reduction of cardiogenic-committed precursors identified as Sca1+/αMHC+ (0.8±0.2% vs. 1.8±0.1%) and Tbx5+ (3.5±0.3% vs. 5.2±0.5%). In contrast, endothelial Sca1+/CD31+ cells were significantly higher in KO mice (11.3±0.4% vs. 8.6±0.4%; n≥9). In addition, Sca1+ isolated cells of Klf15 KO showed increased RNA expression of endothelial markers von Willebrand Factor, CD105, and Flk1 along with upregulation of β-catenin target genes.

CPCs co-cultured on adult fibroblasts resulted in increased endothelial Flk1 cells and reduction of αMHC and Hand1 cardiogenic cells in KO vs. WT CPCs (n=9). Treating these co-cultures with Quercetin, an inhibitor of nuclear β-catenin, resulted in partial rescue of the observed phenotype.

This study uncovers a critical role of KLF15 for the maintenance of cardiac tissue homeostasis. Via inhibition of β-catenin transcription, KLF15 controls cardiomyogenic cell fate similar to embryonic cardiogenesis. This knowledge may provide a tool for activation of endogenous CPCs in the postnatal heart.
Abstract 358: Impaired Function of Circulating Cd34
+
Cd45
-
Cells in Patients with Persistent Atrial Fibrillation Is Restored by the Calcium Channel Blocker Bepridil Hydrochloride
Authors: Katoh Youichi , Hashimoto Ryota , Yozu Gakuto , Yu-u Hiroshige , Yamase Miki , Enjyoji Yoshihisa , Nakazato Yuji
Published: July 2021

Background:
Despite accumulating evidence regarding the occurrence of endothelial dysfunction in persistent atrial fibrillation (PAF) patients, little is still known about the number and function of circulating endothelial progenitor cells (EPCs) in PAF. Bepridil hydrochloride (BEP) is a multiple ion channel blocker that has exhibited efficacy for the pharmacological conversion of PAF.

Methods and Results:
We evaluated both the number and functional properties of CD34
+
CD45
-
cells as EPCs in 12 consecutive patients with PAF. The number of circulating EPCs was determined by flow cytometry. BEP was administered beginning at a daily dose of 100 mg and was increased up to a maximum of 200 mg for 30 days. Higher levels of circulating CD34
+
CD45
-
cells were observed in patients with PAF in comparison to the control subjects [
0.35
+0.04% vs 0.14
+
0.03% CD34
+
CD45
-
cells in the MNC fraction, p<0.048]. However, the EPCs from patients with PAF exhibited an impaired migration towards SDF-1.

Conclusion:
The number of EPCs was observed to increase, but the EPC function was impaired in patients with persistent AF. BEP may have a beneficial effect on the pharmacological conversion of PAF by helping to restore the EPC function.
Abstract 361: Neuregulin Transcriptional Programming of Embryonic Endothelial Progenitor Cells and Embryonic Stem Cells
Authors: Hao Jijun , Galindo Cristi L , Safa Radwan N , Tran Truc-Linh , Sawyer Douglas B
Published: July 2021

Jijun Hao, Cristi L. Galindo, Radwan N. Safa, Truc-Linh Tran, Douglas B. Sawyer Neuregulin-1 (NRG-1) plays a critical role in heart development by signaling through type I receptor tyrosine kinases in the erbB family (erbB2, erbB3 and erbB4). Mice with disrupted expression of NRG-1, ErbB2, ErbB3 or ErbB4 die
in utero
with failure of cardiac development. We have previously shown that NRG-1 has distinct effects on two embryonic progenitor cell populations that express ErbB2 and ErbB3 receptors. In an embryonic endothelial progenitor cell line (eEPCs) NRG-1 treatment induces phosphorylation of Akt, GSK-3β, and Erk1/2, and protects eEPCs against serum deprivation-induced apoptosis. In embryonic stem cells (ESCs) we find that NRG-1 treatment from day 0∼2 induces cardiomyocyte formation by day 8 in culture, and when ErbB3 is knocked down in the ESCs, NRG-1 fails to promote cardiomyogenesis. To understand early molecular events that might regulate these distinct effects, we analyzed global transcriptional changes induced by NRG-1 in both eEPCs and ESCs using microarrays. There were only 244 significantly differential (p value < 0.05, fold-change > 1.5) genes detected in NRG-1-treated ESCs, while NRG-1 induced differential expression of 1,547 transcripts in eEPCs. Based on functional analysis, the most significantly over-represented function (Fishers Exact Test, p value with FDR < 0.05) in ESCs was “cell morphogenesis during differentiation”. In eEPCs, genes regulated via Ras/MAPK signaling were altered, as were those downstream of the Akt-PI3K pathway and calcium signaling. For both cell lines, the most statistically significant transcription factor identified as a regulator of the genes altered in response to NRG-1 was SRF, consistent with a role for NRG-1 in heart development and regeneration. Based on the results of this study, we constructed a putative signaling pathway whereby NRG mediates cardiomyogenesis in pluripotent stem cells that correlates with phenotypic observations.
Abstract 363: Toll-Like Receptor 3 Activation Promotes Efficient Nuclear Reprogramming and Endothelial Differentiation
Authors: Sayed Nazish , Lee Jieun , Hunter Arwen , Au Kin Fai , Wong Wing , Mocarski Edward , Reijo Pera Renee , Cooke John P
Published: July 2021

Introduction:
Stem cell therapy for vascular regeneration has been investigated using embryonic stem cells. We recently generated endothelial cells (ECs) from human induced pluripotent stem cells (hiPSCs) and investigated their potential to promote the perfusion of ischemic tissue in a murine model of peripheral arterial disease (PAD). However, to utilize iPSCs therapeutically, the cells should be generated via non-integrating approaches to avoid integration of foreign DNA into the genome.

Objective:
The present study highlights underlying mechanisms of reprogramming and investigates the role of novel pathways in enhancing nuclear reprogramming for potential clinical application.

Results:
Since the initial discovery, different non-integrating approaches have been developed to generate iPSCs. One such approach is to deliver the pluripotent factors (Oct4, Sox2, Klf4 and cMyc) as cell-permeant proteins (CPPs). However, human cells have not been reprogrammed using purified CPPs. In seeking to develop this approach, we discovered a striking difference in the pattern of gene expression induced by viral versus protein-based delivery of the reprogramming factors. This suggested that a signaling pathway required for efficient nuclear reprogramming was activated by the retroviral, but not CPP approach. In both gain- and loss-of function studies, we find that activation of toll-like receptor 3 (TLR3) plays a role in the efficient reprogramming of human cells using viral approaches. Stimulation of TLR3 causes rapid changes in the expression of epigenetic modifiers, with chromatin remodeling and changes in gene expression that favors induction of pluripotency. Importantly, knowing that this pathway is critical, we were able to generate human iPSCs using CPPs by adding a TLR3 agonist (Poly IC) to the reprogramming protocol.

Conclusion:
Recognition of the role of innate immunity signaling in reprogramming may advance the therapeutic application of iPSCs. We intend to develop an efficient protein-based system to generate EC and determine their therapeutic potential in animal models of PAD. Furthermore, we have discovered an important signaling pathway in reprogramming, which may have implications in cancer biology and regenerative medicine.
Abstract 365: Targeted Delivery of IGF-1 Following Acute Myocardial Infarction
Authors: Khan Raffay S , Sy Jay C , Brown Milton , Martinez Mario D , Murthy Niren , Davis Michael E
Published: July 2021

During acute myocardial infarction (MI) there is excessive necrosis of myocardial cells, leading to the release of large amounts of DNA, representing a potential target for drug delivery. Hoechst, a commonly used molecule for staining nuclei, binds to the minor groove of double-stranded DNA and can be functionalized to contain reactive groups such as free amines, sulfhydryls, and biotin moieties. Insulin-like growth factor-1 (IGF-1), a small molecule with a short half-life is protective immediately following MI, though there is potential for long-term toxicity and off-target effects. Therefore, we hypothesized that conjugating IGF-1 to Hoechst would increase targeting of IGF-1 to the injured myocardium. Hoechst-IGF1 (H-IGF1) was synthesized by binding Hoechst-biotin to biotinylated IGF-1 via a fluorescent streptavidin linker. Intact cells did not show nuclear staining with H-IGF1, while permeabilized cells had a significant increase in blue fluorescent Hoechst staining, indicating H-IGF1 was cell impermeable but could still bind DNA. Activity of H-IGF1 was demonstrated by Akt phosphorylation in cultured cardiac progenitor cells and was similar to native IGF-1. To determine in-vivo targeting of H-IGF1 to MI, mice underwent 30 minutes of coronary artery ligation followed by reperfusion (I/R). Six hours following MI, mice were injected intravenously with 70ng of H-IGF1, S-IGF1 (streptavidin bound IGF-1 only) or PBS followed by in vivo imaging at 30 and 120 minutes post-injection. At 30 minutes post-injection, we found 3.2% (2.2 of 70ng) of the injected dose of H-IGF1 in infarcted hearts compared with 1.8% (1.3 of 70ng) of S-IGF1 (n=5-7; p<0.05). To confirm that targeting of H-IGF1 was dependent on binding DNA, H-IGF1 pre-bound to double-stranded DNA was injected intravenously after I/R. This led to a significant (p<0.05) decrease in targeted IGF-1 levels. IGF-1 levels determined by ELISA 2 hours post-injection demonstrated a similar trend with increased targeting of H-IGF1 compared with S-IGF1 treated mice (4.2±0.6 ng vs. 2.4±0.2 ng; p<0.05). In conclusion, our data demonstrate that intravenous delivery of Hoechst-conjugated IGF-1 increases myocardial targeting. This provides a novel strategy for delivery of growth factors for the treatment of MI.
Abstract 368: Structural and Molecular Mechanisms of Excitation-contraction Uncoupling in Heart Failure
Authors: Xu Ming , Wu Hao-Di , Li Rong-Chang , Li Su-Fang , Lai Shao-Ting , Zhang You-Yi , Wang Shi-Qiang
Published: July 2021

The excitation-contraction (E-C) coupling in heart cells governed by L-type Ca
2+
channels (LCCs) in the cell membrane/T-tubules (TTs) and ryanodine receptors (RyRs) in the junctional sarcoplasmic reticulum (JSR). During heart failure, LCC-RyR signaling became defective, leading to degraded Ca
2+
transients and compromised contractile strength. In the present study, we investigated the structural and molecular mechanisms underlying the compromised LCC-RyR signaling in rat heart failure models produced by transverse aortic constriction surgery. Stereological analysis of transmission electron microscopic images showed that the volume density and the surface area of JSRs and those of JSR-coupled TTs were both decreased in failing heart cells. The TT-JSR junctional structures, known as couplons, were displaced or missing from the Z-line areas. Moreover, the spatial span of individual couplons was markedly reduced in failing heart cells. Knockdown of junctophilin-2 (JP2), a TT-SR linker protein down-regulated in heart failure, fully reproduced the structural deformation and decreased/desynchronized Ca
2+
release, indicating that JP2 down-regulation is at least partially responsible for the structural and functional defects of E-C coupling in heart failure. In searching for upstream regulator of JP2, we found that microRNA-mediated suppression of JP2 expression underlied both TT-SR structural uncoupling and LCC-RyR functional uncoupling. These results revealed molecular and structural mechanisms underlying the defective E-C coupling in failing heart cells, and suggested new therapeutic targets against heart failure.
Abstract 370: Malignant Hyperthermia Mutation of RyR1 (Y522S) Increases Catecholamine-Induced Cardiac Arrhythmia Through Mitochondrial Injury
Authors: O-Uchi Jin , Porter George A , Kang Sung Hyun , Boncompagni Simona , Sokolova Niina , Gross Polina , Jhun Bong Sook , Beutner Gisela , Brookes Paul , Blaxall Burns C , Dirksen Robert T , Protasi Feliciano , Pan Shi , Sheu Shey-Shing
Published: July 2021

Introduction:
Mutations in skeletal muscle type ryanodine receptor (RyR1) exhibit life-treating human disorders including malignant hyperthermia (MH) and central core disease, which features skeletal muscle rigidity triggered by anesthesia and body temperature elevation. However, cardiac arrhythmias and/or sudden cardiac death in these patients are also frequently reported during anesthesia or even under the conscious condition.

Hypothesis:
Chronic mitochondrial Ca
2+
overload through leaky mutant RyR1 expressed at cardiac mitochondria leads to mitochondria injury and cardiac arrhythmia.

Methods:
We determined the cardiac phenotype of knock-in mice carrying a leaky RyR1 MH mutation Y522S (YS).

Results:
WT- and YS
-
RyR1 were expressed at mitochondria but not at SR in heart. Ultra-structure of heterozygous YS hearts exhibited severe abnormal/disrupted mitochondrial morphology as well as myofibril contractures (Fig. A-E). YS cardiac mitochondria showed high susceptibility to Ca
2+
-induced opening of mitochondrial permeability transition pores and depolarized mitochondrial membrane potential compared to WT, suggesting compromised mitochondrial functions. Langendorff perfusion developed similar LV pressure in response to high dose bolus of Isoproterenol in WT and YS, but only YS heart frequently developed multiple ventricular extrasystoles after Isoproterenol (Fig. F).

Conclusion:
Chronic mitochondrial damage by Ca
2+
overload through leaky mutant RyR1 induces mitochondrial structural and functional disruption, which facilitates arrhythmogenic outbursts under acute catecholaminergic stress.
Abstract 49: Prolyl Isomerase Pin1 Regulates Cardiac Hypertrophy via Controlling Phosphorylation of Akt and MEK
Authors: Toko Haruhiro , Konstandin Mathias , Gude Natalie , Sussman Mark
Published: July 2021

Rationale:
Kinases and phosphatases regulate crucial aspects of growth and survival through phosphorylation and dephosphorylation of target substrates. Processes of cardiac hypertrophy, myocardial infarction, and heart failure are dictated in part by which kinases or phosphatases are involved and also by the intensity and duration of specific enzymatic activities. While research has identified numerous critical regulatory kinases and phosphatases in the myocardium, the intracellular mechanism for temporal regulation of signaling duration and intensity remains obscure. In the non-myocyte context, control of folding, activity, stability, and subcellular localization of proteins responsible for growth and survival is mediated by the prolyl isomerase Pin1.

Objective:
To establish the role of Pin1 in the heart.

Method and Results:
Initial characterization of myocardial Pin1 involved assessment of expression and localization during postnatal development or pathological challenge. Pin1 protein level was decreased and the location of Pin1 was changed from nucleus to cytoplasm with increasing age. Next, Pin1 protein expression and localization were assessed in the pathological challenged heart. Pin1 protein expression increases with pressure overload and ischemia, particularly in perivascular areas and in border zone myocytes, respectively. To determine the role of Pin1 on cardiac hypertrophy, siRNA to Pin1 (siPin1) was applied to neonatal rat cardiomyocytes. Western blot analysis showed that siPin1 decreased phosphorylation of Akt, and immunohistochemical analysis showed that siPin1 reduced cardiomyocyte size in response to high serum. siPin1 also decreased phosphorylation of MEK and reduced cardiomyocyte size in response to phenylephrine treatment. Furthermore, cardiac hypertrophy induced by transaortic constriction was ameliorated in Pin1 knockout mice, compared with littermate wild type mice.

Conclusion:
Expression and location of Pin1 during development and in response to pathologic challenge point to an important role for Pin1 in adaptation to myocardial growth or stress. Collective evidence indicates that Pin1 controls cardiac hypertrophy at least in part via regulating phosphorylation of Akt and MEK.
Abstract 52: Impaired Ephrin A1/EphA2 Signaling Results in Defective Migration and Homing of Senescent Human Cardiac Stem Cells
Authors: Kannappan Ramaswamy , Bai Yingnan , Signore Sergio , Cimini Maria , Ferreira-Martins Joao , Goichberg Polina
Published: July 2021

Aging is the major independent risk factor for chronic heart failure. Despite the presence of cardiac stem cells (CSCs), the old human heart undergoes progressive deterioration in ventricular performance, coupled with scattered foci of fibrosis and accumulation of poorly contracting myocytes. We raised the possibility that defects in the translocation of senescent human CSCs (hCSCs) to the sites of damage constitute a key determinant in the manifestation of the aging myopathy. We report that ephrin A1-EphA2 receptor signaling is a critical modulator of hCSC motility. Ephrin A1, a membrane-anchored protein, is expressed on the myocyte sarcolemma and acts as a ligand for the EphA2 receptor on neighboring hCSCs, facilitating their migration. Pre-treatment of young hCSCs with ephrin A1 resulted in enhanced movement of the transplanted cells to the necrotic tissue, with formation of new myocardium and improvement in cardiac function. Whether senescent hCSCs promote a comparable regenerative response remained to be established. Surprisingly, the expression of EphA2 did not differ in young and old hCSCs. With respect to young cells, senescent hCSCs showed a 2-fold increase in intracellular ROS levels. Oxidative stress led to post-translational modifications and functional alterations of the EphA2 receptor. Specifically, the ability of ephrin A1 to induce phosphorylation of the EphA2 receptor was markedly attenuated in senescent hCSCs, resulting in inadequate activation of Src family proteins. As a consequence, the phosphorylation and activity of caveolin-1, a substrate of Src kinases, was reduced. These molecular alterations led to impaired endocytosis of the ligand-receptor complex, a cellular process essential for ephrin A1-EphA2 signaling. Lack of endocytosis precluded rearrangement of the actin cytoskeleton and cell migration. Importantly, ephrin A1-stimulated senescent hCSCs delivered to infarcted rats accumulated in proximity of the site of injection and did not translocate to the ischemic area. Thus, oxidative stress interferes with EphA2 signaling in aging hCSCs, negatively affecting their migration. Restoration of the EphA2 function in old hCSCs may enhance their mobilization and improve cell targeting to the injured area.
Abstract 54: Probenecid Improves Myocardial Function in an Ischemic Heart Disease Murine Model
Authors: Robbins Nathan , Koch Sheryl E , Jiang Min , Tranter Michael , Ren Xiaoping , Jones W Keith , Rubinstein Jack
Published: July 2021

Introduction
Probenecid, previously used for the treatment of gout, is a transient receptor potential vanilloid 2 (TRPV2) agonist. We have found TRPV2 in murine cardiomyocytes and when stimulated by probenecid, results in a positive inotropic response through Ca
2+
influx independent of β-adrenergic signaling. Our
hypothesis
is that probenecid will increase contractility without affecting cell survival.

Methods
We studied the role of probenecid as a positive inotrope in a mouse model of ischemic heart disease. We administered probenecid via intraperitoneal injections (100mg/kg) before and after ischemia/reperfusion injury (I/R) and orally via treated water (100mg/kg daily for 4 weeks), using saline injections and untreated water as respective controls. Ischemia was induced by a 45 minute ligation of the LAD followed by reperfusion and all mice were followed serially via echocardiography. We also evaluated the effects of probenecid on HL-1 cell apoptosis by conducting cell viability assays.

Results
Treatment with probenecid before I/R had no effect on subsequent infarct size (51.6±3.71% vs. 53.4±1.54%;P=NS). However, post-I/R probenecid caused an increase in ejection fraction (EF) of 10.9±1.98% in mice with initial EF between 40-50% (n=6; P<0.01) and 7.28±1.63% in mice with higher initial EF (50-60%) (n=6; p<0.01). Echocardiographic analysis of mice with oral probenecid after I/R demonstrated higher EF and smaller diastolic volume (47.19±2.29%; 79.57mL±1.63; n=6) compared to untreated mice (43.67±2.91%; 89.05mL±5.67; n=6). Cell viability assays showed high concentrations of probenecid had minimal effect on cell viability (88.6±6.2% of control) whereas treatment with high concentrations of isoproterenol (10mM) greatly decreased cell viability (6.7±0.1% of control).

Conclusions
These experiments demonstrate 1) probenecid increases myocardial contractility at baseline and significantly more so after I/R injury; 2) probenecid therapy improves function when used chronically; 3) the increase in contractility is not associated with either increased infarct size in vivo or apoptosis in vitro. Thus, unlike other positive inotropes, such as isoproterenol, probenecid increases contractility without resulting in significant cell death.
Abstract 64: Meis1 Is a Key Regulator of Postnatal Cardiomyocyte Cell Cycle Arrest
Authors: Kocabas Fatih , Mahmoud Ahmed I , Muralidhar Shalini , Porrello Enzo , Olson Eric , Sadek Hesham A
Published: July 2021

Heart failure is a costly and deadly disease affecting over 5 million Americans. The inability of the adult mammalian heart to regenerate following injury lies at core of the pathophysiology of heart failure. We recently showed that the neonatal mammalian heart is capable of complete regeneration following resection of the entire ventricular apex. Moreover, our preliminary data indicate that the neonatal mouse heart is also capable of complete regeneration following ischemic myocardial infarction. In both these types of injury, the regenerative response is associated with robust proliferation of cardiomyocytes without significant hypertrophy or fibrosis. Genetic fate mapping studies demonstrated that the majority of newly formed cardiomyocytes originated from pre-existing cardiomyocytes. In an effort to determine the mechanism of cardiomyocytes cell cycle arrest after the first week of life, we performed a gene array after cardiac injury at multiple post-natal timepoints. This enabled us to identify Meis1 as a potential regulator of neonatal cardiomyocyte proliferation. Meis1, which belongs to the TALE family of homeodomain transcription factors, is required for normal hematopoiesis and cardiac development. While Meis1 has been extensively studied in the hematopoietic system, little is known about its role in the heart. Our results indicate that Meis1 expression and nuclear localization in the post-natal cardiomyocytes coincides with cell cycle arrest. To further explore this pattern, we generated a cardiomyocyte-specific Meis1 knockout mouse, and showed that loss of Meis1 results in robust cardiomyocyte proliferation in the adult heart. Moreover, we identified p16 and p21; two synergistic cyclin dependent kinase inhibitors that induce arrest at all three cell cycle checkpoints, as potential targets for transcriptional activation by Meis1. These results identify Meis1 as a key regulator of post-natal cardiomyocyte cell cycle arrest, and a potential therapeutic target for cardiac regeneration.
Abstract 65: β-Adrenergic Signaling Promotes Survival and Proliferation of Mouse Cardiac Progenitor Cells Prior to Lineage Commitment
Authors: Khan Mohsin , Mohsin Sadia , Avitabile Daniele , Nguyen Jonathan , Gude Natalie , Sussman Mark A
Published: July 2021

Rationale:

Short term β-adrenergic stimulation promotes contractility in response to stress, but is ultimately detrimental in the failing heart due to accrual of cardiomyocyte death. Endogenous myocardial repair may partially offset cardiomyocyte losses, but consequences of long term β-adrenergic drive upon myocardial repair and regeneration are unknown.

Objective:

Modest recovery of cardiac contractility following long term β-adrenergic blockade in the clinical setting may depend, in part, upon restoration of endogenous repair therefore we sought to determine the relationship between β-adrenergic activity and regulation of cardiac progenitor cell (CPC) function and influence upon myocardial repair.

Methods and results:

Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-AR signaling promotes proliferation associated with increased Akt phosphorylation, up-regulation of eNOS and cyclin D1, and decreased levels of GRK2. Conversely, silencing of β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by the β1-AR specific antagonist metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed
in vivo
by adoptive transfer of CPC into failing mouse myocardium, concomitant with increased ejection fraction, fractional shortening and hemodynamic performance.

Conclusions:

β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR upon commitment to the myocyte lineage results in loss of early myocyte precursors and ineffective myocardial repair. Thus, β1-AR-specific blockade is likely to provide for enhanced CPC participation in recovery of function in the failing heart.
Abstract 7: S-nitrosylation Affects Differentiation of Mesenchymal Stem Cells
Authors: Cao Yenong , Gomes Samirah A , Rangel Erika B , Paulino Ellena C , Kulandavelu Shathiyah , Balkan Wayne , Hare Joshua M
Published: July 2021

Introduction:
Bone marrow-derived mesenchymal stem cells (BMMSCs) appear to play an important role in the formation of atherosclerotic lesions. Circulating BMMSCs are attracted to arterial wall where they undergo adipogenic or osteogenic differentiation to form ectopic aggregates of fat and bone. The factors that determine the direction of BMMSC differentiation are poorly understood. We assessed the role of nitric oxide (NO), an inhibitor of lipid formation and vascular calcification, in BMMSC differentiation.

Hypothesis:
S-nitrosylation mediated-NO signaling controls the balance between adipogenic and osteogenic differentiation of BMMSCs.

Methods:
We isolated BMMSCs from wild type mice (WT) and mice deficient in S-nitrosoglutathione reductase (GSNOR
-/-
), an enzyme that governs levels of S-nitrosylation by promoting protein denitrosylation. Cells were cultured in either adipogenic or osteogenic differentiation media followed by functional and gene expression assays.

Results:
MSCs derived from GSNOR
-/-
mice had impaired fat droplet formation and lower expression of the adipogenic markers PPARγ (1329±415.3-fold increase in WT vs. 158±65.61-fold in GSNOR
-/-
, P<0.05) and FABP4 (11.06±3.29-fold in WT vs. 4.06±0.62-fold in GSNOR
-/-
, P<0.05). Conversely, GSNOR
-/-
MSCs exhibited enhanced osteogenic differentiation as indicated by greater calcium deposition and higher expression of the osteogenic marker Osteopontin (1.48±0.17-fold in WT vs. 16.18±5.26-fold in GSNOR
-/-
, P<0.05). Interestingly, GSNOR
-/-
cells had higher baseline expression of Osteopontin, Osteocalcin and Runx2. Treatment of GSNOR
-/-
MSCs with L-NAME, a nitric oxide synthase (NOS) inhibitor, resulted in decreased osteogenic differentiation without affecting adipogenic differentiation.

Conclusion:
Our results indicate that GSNOR enhances adipogenic differentiation and impairs osteogenic differentiation of BMMSCs
in vitro
and suggest that interaction of GSNOR- and NOS-mediated processes regulates the balance between adipogenic and osteogenic differentiation of BMMSCs.
Abstract 74: Naturally Occurring Human α1a-Adrenergic Receptor Genetic Variant Induces Transformation of Cardiomyoblasts to Fibroblast-Like Phenotype
Authors: Kleine-Brueggeney Maren , Gradinaru Irina , Babaeva Ekaterina , Schwinn Debra A , Oganesian Anush
Published: July 2021

Hypertensive heart disease is a progressive condition leading to myocardial remodeling, alterations in cardiomyocytes and fibrosis. Three α1 adrenergic receptor (AR) subtypes are expressed in human heart (α1a, α1b, α1d). Recently, we reported that human α1aAR with the naturally occurring G247R SNP (247R), originally identified in a hypertensive patient, constitutively couples to EGFR-transactivation pathway in fibroblasts and leads to hyperproliferation. We hypothesized that expression of 247R in cardiomyoblasts plays an important role in their remodeling.

We demonstrate that constitutive expression of 247R in H9C2 cardiomyoblasts changes the morphology of myoblasts to fibroblast-like cells (transformed, tr247R); higher 247R expression results in earlier onset of transformation. Expression of α1aAR-WT does not affect cell morphology. Tr247R cells exhibit 2-3 fold increased constitutive proliferation compared to control or WT cells as determined by cell counts and thymidine incorporation assays; pERK is upregulated in tr247R cells as determined by Western blot analyses. Treatment with AraC, a DNA replication inhibitor, eliminates the 247R cells but not the H9C2 myoblasts. Expression of cell-specific markers is being evaluated in tr247R and control cells. RT-PCR and zymograms revealed increased MMP2 mRNA and protein levels in tr247R cells compared to control or WT cells and are comparable to MMP2 levels in fibroblasts. Increased proliferation of tr247R cells is reduced by EGFR specific inhibitor AG1478 but not general MMP inhibitor GM6001. Agonist stimulation of tr247R results in hypertrophy as determined by leucine incorporation. Interestingly, hypertrophy but not cell proliferation is inhibited by prazosin, an α1aAR inverse agonist, suggesting that increased proliferation and hypertrophy observed in transformed cells are two independent pathways triggered by 247R.

Our data demonstrate that expression of α1aAR genetic variant triggers alterations in cardiomyoblasts via distinct signaling pathways leading to hyperproliferation, transformation of the cells to fibroblast-like phenotype and hypertrophy. Consequently, these changes may lead to fibrosis in human heart, extracellular matrix remodeling and heart failure.