bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 356: Scl Represses Cardiogenesis via Distant Enhancers during Hemogenic Endothelium Specification
Authors: Org Tonis , Duan Dan , Ferrari Roberto , Montel-Hagen Amelie , Van Handel Ben , Sasidharan Rajkumar , Orkin Stuart H , Kurdistani Siavash , Mikkola Hanna K
Published: July 2021

Understanding the mechanisms directing mesoderm specification holds a great potential to advance the development of cell-based therapies for cardiovascular and blood disorders. The bHLH transcription factor Scl is known as the master regulator of the hematopoietic fate. We recently discovered that, in addition to its critical function in promoting the establishment of hemogenic endothelium during hematopoietic stem/progenitor cell (HS/PC) development, Scl is also required to repress cardiomyogenesis in endothelium in hematopoietic tissues and endocardium in the heart. However, the mechanisms for the cardiac repression have remained unknown.

Using ChIP-sequencing and microarray analysis of Flk+ mesoderm differentiated from mouse ES cells, we show that Scl both directly activates a broad gene regulatory network required for hemogenic endothelium and HS/PC development (e.g. Runx1, cMyb, Lyl1, Mef2C, Sox7 etc.), and directly represses transcriptional regulators required for cardiogenesis (e.g. Gata4, Gata6, Myocd, etc.) and mesoderm development (Eomes, Mixl1, Etv2, etc.). Repression of cardiac and mesodermal programs occurs during a short developmental window through Scl binding to distant enhancers, while binding to hematopoietic regulators extends throughout HS/PC and red blood cell development and encompasses both distant and proximal binding sites. We also discovered that, surprisingly, Scl complex partners Gata 1 and 2 are dispensable for hematopoietic vs. cardiac specification and Scl binding to majority of its target genes. Nevertheless, Gata factors co-operate with Scl to activate selected transcription factors that facilitate HS/PC emergence from hemogenic endothelium. These results denote Scl as a true master regulator of hematopoietic vs. cardiac fate choice and suggest a mechanism by which lineage-specific bHLH factors direct the divergence of competing fates.
Abstract 10: Cardiac-specific Yap Activation Improve Cardiac Function And Survival In An Experimental Murine Mi Model
Authors: Lin Zhiqiang , von Gise Alexander , Zhou Pingzhu , Ma Qing , Chen Jinghai , Jiang Jianming , Seidman Jonathan G , Wang Da-zhi , Pu William T
Published: July 2021

Background:
Yes-Associated Protein (YAP), the terminal effector of the Hippo signaling pathway, is crucial for regulating embryonic cardiomyocyte proliferation. We hypothesized that YAP activation after myocardial infarction would preserve cardiac function and improve survival.

Methods and Results:
In this study, we used a cardiac-specific, inducible expression system to activate YAP in adult mouse heart. Activation of YAP in adult heart promoted cardiomyocyte proliferation and did not deleteriously affect heart function. Furthermore, YAP activation after myocardial infarction (MI) preserved heart function and reduced infarct size. Using adeno-associated virus subtype 9 (AAV9) as a delivery vector, we expressed human YAP in the adult murine myocardium immediately after MI. We found that AAV9:hYAP significantly improved cardiac function and mouse survival. AAV9:hYAP did not exert its salutary effects by reducing cardiomyocyte apoptosis. Rather, AAV9:hYAP stimulated adult cardiomyocyte proliferation. Gene expression profiling indicated that AAV9:hYAP stimulated cell cycle gene expression, activated of components of the inflammatory response, and promoted a less mature cardiac gene expression signature.

Conclusions:
Cardiac specific YAP activation after MI mitigated myocardial injury, improved cardiac function, and enhanced survival. These findings suggest that therapeutic activation of YAP or its downstream targets, potentially through AAV-mediated gene therapy, may be a strategy to improve outcome after MI.
Abstract 102: Acute Mechanical Unloading of the Left Ventricle Promotes Cardioprotective Signaling and Limits Myocardial Infarct Size
Authors: Kapur Navin K , Paruchuri Vikram , Qiao Xiaoying , Morine Kevin , Syed Wajih , Salehi Hamid , Dow Sam , Shah Nimish , Esposito Michele , Pandian Natesa , Karas Richard H
Published: July 2021

Management of an acute myocardial infarction (AMI) focuses on restoring oxygen supply to limit myocardial damage, however ischemia-reperfusion injury (IRI) remains a major determinant of mortality in AMI. No studies have targeted initially reducing left ventricular stroke work (LVSW) to limit IRI in AMI. The Impella CP axial-flow pump reduces LVSW. We tested the hypothesis that first reducing myocardial work and delaying coronary reperfusion reduces infarct size by activating cardioprotective signaling pathways.

Methods:
AMI was induced by occlusion of the left anterior descending artery (LAD) via angioplasty for 90 minutes in 50kg male Yorkshire swine (n=5/group). In Group 1, the LAD was reperfused for 120 minutes. In Group 2, after 90 minutes of ischemia the Impella CP device was activated and the LAD left occluded for an additional 60 minutes (150 minutes of LAD occlusion total), followed by 120 minutes of reperfusion. The Impella CP was active throughout reperfusion. Western blot analysis quantified myocardial kinase activity.

Results:
Compared to Group 1, Group 2 had a reduced LVSW, LV end-diastolic volume and end-diastolic pressure after reperfusion [Fig A]. Group 2 showed increased myocardial phosphorylation of cardioprotective kinases: AKT, ERK, GSK3β and STAT-3 [Fig B]. Compared to Group 1, the percent myocardial infarct size normalized to the area at risk (AAR) was reduced in Group 2 (73+13% vs 42+15%, p=0.02).

Conclusion:
We report the potential benefit of primarily unloading the heart and delaying coronary reperfusion to salvage myocardium in AMI. This is the first report to examine the impact of the Impella CP on cardioprotective signaling in the heart.
Abstract 103: Interferon Regulatory Factor-1 Is Responsible For Cardiac Protective Effect Of 5-azacytidine On Macrophages In Myocardial Infarction
Authors: Kim Yong Sook , Jeong Hye-yun , Ahn Youngkeun
Published: July 2021

Background:
During progression of cardiac injury, macrophages with specialized phenotypes are actively involved in inflammatory responses. In this study, we investigated the effect of 5-azacytidine (5AZ) on macrophages in the damaged myocardium.

Methods:
A mouse macrophage cell line RAW264.7 was stimulated with lipopolysaccharide (LPS, 100 ng/mL) with or without 5AZ (10 μM). Nitric oxide was quantified by Griess’s method. Expression patterns of inducible nitric oxide synthase (iNOS) and interferon regulatory factor 1 (IRF-1) were determined by Western blot analysis. For animal study, myocardial infarction (MI) was induced by ligation of left coronary artery in mice, and divided into four groups; non MI + saline, non-MI + 5AZ , MI + saline, and MI + 5AZ. Saline or 5AZ was injected (5 mg/kg/d) every other day. Cardiac fibrosis was evaluated by Masson’s trichrome stain was performed and cardiac function was by echocardiography 2 weeks after MI.

Results:
LPS-induced nitric oxide formation was reduced by 5AZ treatment in RAW264.7 cells. LPS-induced iNOS mRNA and protein inductions were blocked by 5AZ treatment. Next, the effect of 5AZ on IRF1, a regulator of iNOS, was examined. IRF-1 was dramatically increased by LPS to peak at 4 hr and then reduced to basal level. In the presence of MG132, a proteasome inhibitor, IRF-1 protein sustained maximal level without degradation. On the other hand, IRF-1 protein was significantly highly maintained by 5AZ treatment. In animal study, there were significant improvements in ejection fraction (53.25 ± 2.55 % vs. 62.50 ± 7.2 %, p < 0.05) and cardiac fibrosis (28.85 ± 5.44% vs. 16.57 ± 6.30%, p < 0.05). Collectively, 5AZ inhibited iNOS induction by modulation of IRF-1 in the activated macrophages to preserved cardiac function and fibrosis after MI.

Conclusion:
5AZ protected post-MI injury by regulation of IRF-1 kinetics to modulate macrophages in infarcted myocardium. This study suggested 5AZ as a novel therapeutic intervention for cardiac repair.
Abstract 105: Downregulation Of Cardiac Na+ Channel In Myocardial Infarction Is Prevented By A Mitochondria-targeted Antioxidant
Authors: Liu Man , Rutledge Cody A , Yang Kai-Chien , Dudley Samuel C
Published: July 2021

Objectives:
The aim of this study was to investigate the effect of ischemia on cardiac Na+ channel (Nav1.5) and possible treatments in a mouse model of myocardial infarction (MI).

Methods:
MI was induced in 12-week old C57BL/6 mice by coronary artery occlusion. Sham-operated mice were used as controls. Two weeks following surgery, MI mice were either given a mitochondria-targeted antioxidant, mitoTEMPO (0.7 mg/kg/day, intraperitoneally), or left untreated for two weeks. Cardiomyocytes isolated from the scar border of MI mice or from the left ventricular (LV) anterior wall of sham-operated mice were utilized for whole-cell patch clamp recording of Na+ currents (INa) and for measurements of mitochondrial reactive oxygen species (mitoROS) using flow cytometry. Nav1.5 protein expression levels were determined in the LV from MI and sham-operated mice. Echocardiography was performed 2- and 4-weeks following MI.

Results:
The peak INa densities of the isolated LV cardiomyocytes were significantly lower (P<0.05) in MI (-14.3±1.4 pA/pF), compared to sham (-24.0±1.8 pA/pF). The mitoROS levels were elevated to 1.5±0.2 fold in MI mice (P<0.05). INa was increased (-19.4±0.8 pA/pF, P<0.05) and mitoROS was decreased to 1.2±0.2 fold (P<0.05) with mitoTEMPO treatment. The Nav1.5 channel protein level was not altered in the heart tissue of MI mice. There were no significant differences in echocardiography parameters between untreated and mitoTEMPO groups to explain the increase in INa.

Conclusions:
Ischemic cardiomyopathy leads to downregulation of cardiac Nav1.5 currents and overproduction of mitochondrial ROS. The mitochondria-targeted antioxidant can mitigate these changes and may help reduce arrhythmic risk after myocardial infarction.
Abstract 106: GYY4137 Attenuates Post-Ischemic Adverse Remodeling and Modulates ANP-Mediated cGMP Signal Transduction
Authors: Martinez Eliana C , Lilyanna Shera , Peh Meng T , Liew Oi W , Moore Philip K , Richards Arthur M
Published: July 2021

Myocardial infarction is the most frequent proximate cause of heart failure, which remains a leading cause of death in the developed world. Hydrogen sulfide (H
2
S) is emerging as an important endogenous modulator in diverse physiological and pathophysiological events. We hypothesized that the slow-releasing water-soluble H
2
S donor GYY4137 (GYY) may exert cardioprotective effects through modulation of neurohormonal response to cardiac injury. We have found that treatment for 1 week with GYY (100mg/Kg/48hr, IP) after acute myocardial infarction in rats, provides powerful sustained preservation of left ventricular (LV) dimensions and function in vivo, compared to untreated infarcted (MI), placebo- and D-propargylglycine- (PAG, an inhibitor of endogenous H
2
S synthesis) treated animals 2 and 7 days after infarct (n=6/group/time-point). LV dimensions and function in GYY-treated animals were comparable to healthy sham-operated rats. GYY-treated hearts displayed a significantly lower percentage of LV fibrosis than MI, placebo and PAG hearts, whereas PAG treated animals had significantly bigger scar size relative to GYY, at days 2 and 7 after MI. A higher density of blood vessels was found in the scar area of GYY-treated animals compared to all other infarcted groups at days 2 (P<0.0002) and 7 (P<0.01) post-MI. Furthermore, treatment with GYY resulted in increased levels of plasma atrial natriuretic peptide (ANP) compared to all groups at days 2 and 7 after MI, while cardiac mRNA expression of ANP was also significantly increased in GYY-treated rats compared to Sham (P<0.001), and all the infarcted groups (P<0.05) at day 2 after MI. Concordantly, ANP second messenger cGMP was increased in plasma at day 7 in GYY rats compared to sham and all infarcted groups (p<0.05), paralleled by higher cGMP-dependent protein kinase type I (cGKI) protein levels than sham (P<0.01), and vasodilator-stimulated phosphoprotein phosphorylation (pVASP) at the cGKI preferred site (Ser239) compared to sham and PAG (P<0.05) at day 2 post-MI. In conclusion, our data suggest that H2S attenuates adverse remodeling and may exert post-ischemic cardioprotective (pro-angiogenic, anti-fibrotic) effects in part through modulation of ANP-mediated cGMP signal transduction.
Abstract 109: Interleukin 17 A, Coronary Artery Disease And Gender of The Patients
Authors: Oliveira Dinaldo C , Heide Elayne , Rego Moacyr , Oliveira Danielle A , Sarinho Felipi W , Gomes Viviane R , Pita Maira G
Published: July 2021

Introduction:
The effects of interleukins during development of coronary artery disease (CAD) have been studied by researchers and it may help to understand better this disease. The aim of this study was to evaluate if there are differences of concentrations of the IL 17 A between men and women with stable CAD.

Hypothesis:
There are no differences of concentrations of the IL 17 A according to gender of the patients.

Methods:
This is a cross-sectional, prospective, analytical study, conducted from August to December 2012. We included 40 patients (24 men (MP) and 16 women (WP) with stable CAD, CCS III or IV, ischemic myocardial scintigraphy, who had not been subjected to any kind of myocardial revascularization and with coronary stenosis ≥ 50% according to current coronary angiography. There were 20 healthy volunteers (12 men (MC) and 8 women (WC), to take up comparison of concentrations of interleukins (IL). Interleukins were evaluated in serum of patients and after 48 hours of cells in culture with and without stimulus. IL concentrations were expressed in pg / ml. Statistical analysis was performed using the Mann-Whitney or Student t test. P ≤ 0,05 was considered statistically significant.

Results:
The comparison between the groups showed:

IL 17 A: Serum: MP = 3.91 (3.91 - 3.91) vs MC = 3.91 (3.91 - 9.28), p = 0.31; culture 48 hours without stimulus: MP = 3.91 (3.91 - 3.91) vs MC = 3.91 (3.91 - 3.91), p = 0.45; culture 48 hours with stimulus: MP = 451.67 (99.02 - 892.58) vs MC = 135 (3.91 -- 285), p = 0.04.

IL 17 A: Serum: WP = 3.91 (3.91 - 3.91) vs WC = 3.91 (3.91 -- 6.25), p = 0.90; culture 48 hours without stimulus: WP = 3.91 (3.91 - 3.91) vs WC = 3.91 (3.91 -- 3.91), p = 0.20; culture 48 hours with stimulus: WP = 131.21 (3.91 - 231.97) vs WC = 173.78 (3.91 - 642), p = 0.24.

IL 17 A: Serum: MP = 3.91 (3.91 - 3.91) vs WP = 3.91 (3.91 - 3.91), p = 0.17; culture 48 hours without stimulus: MP = 3.91 (3.91 - 3.91) vs WP = 3.91 (3.91 - 3.91), p = 0.47; culture 48 hours with stimulus: MP = 451.67 (99.02 -- 892.58) vs WP = 131.21 (3.91 - 231.97), p = 0.02.

Conclusions:
There were no differences of concentrations of the IL 17 A between WP and WC. However, MP had higher concentrations of the IL 17 A than MC and WP. Therefore, it is possible that the IL 17 A take part in a pathway which determines different patterns of CAD according to gender.
Abstract 11: Xenogen-free In Vitro Differentiation and Expansion of Human Pluripotent Stem Cell-Derived Cardiovascular Progenitor Cells
Authors: Meng Xianmei , Knopf Anne , Vega-Crespo Agustin , Byrne James , Van Handel Ben , Nsair Ali
Published: July 2021

Background:
Human pluripotent stem cell-derived cardiovascular progenitor cells (hPSC-CPCs) represent a tractable option for cell-based therapy for heart disease. However, to be clinically relevant, these cells must be derived under good manufacturing practices (GMP)-compatible conditions and produced in great enough quantities to treat adult patients. Here we sought to demonstrate for the first time the generation and expansion of clinically relevant numbers of hPSC-CPCs in xenogen-free protocol.

Methods and Results:
GMP-grade human induced pluripotent stem cells (GMP-hiPSCs) and human embryonic stem cells (H1 and H9) were dissociated into single cells and cultured in low attachment dishes to differentiate into CPCs in StemPro medium including small molecules and human cytokines with high efficiency of 86%, 80% and 66% for GMP-hiPSCs, H1 and H9, respectively (Figure 1). All hPSC-CPCs possessed trilineage differentiation potentials, as shown by differentiation into endothelial and smooth muscle cells and functional cardiomyocytes (Figure 2). Moreover, sorted hPSC-CPCs expanded >5 fold in 10 days in xenogen-free conditions while still maintaining trilineage differentiation potential and an efficiency of ~70% (Figure 3).

Conclusions:
Here we demonstrate a xenogeny-free CPC derivation and expansion protocol that can generate clinically relevant numbers of GMP-grade cardiovascular progenitors that could be used in a clinical setting.
Abstract 114: Cytokine Response to Diet and Exercise Affects Atheromatous Matrix Metalloproteinase-2/9 Activity in Mice
Authors: Kim Dong-Eog
Published: July 2021

Objective:
To identify the principal circulating factors that modulate atheromatous matrix metalloproteinase (MMP) activity in response to diet and exercise.

Methods and Results:
Apolipoprotein-E knock-out mice (n=56) with preexisting plaque, fed either Western diet (WD) or normal diet (ND), underwent either 10-week treadmill exercise or not. In vivo atheromatous MMP activity was visualized using molecular imaging with an MMP-2/9 activatable near-infrared-fluorescent probe. We measured atherosclerosis-related cytokines, lipid levels, visceral fat, and correlated these outcome measures to atheromatous MMP activity. Body weight, visceral fat, and plaque size were all higher in WD-fed animals than in ND-fed animals. Exercise training did not significantly affect these parameters in either WD-fed animals or ND-fed animals. However, atheromatous MMP activity was different: ND animals with and without exercise had similar low MMP activities, WD animals without exercise had high MMP activity, and WD animals with exercise had reduced levels of MMP activity, close to the levels of ND animals. Factor analysis and path analysis showed that soluble vascular cell adhesion molecule (sVCAM)-1 was directly positively related to atheromatous MMP activity. Adiponectin was indirectly negatively related to atheromatous MMP activity by way of sVCAM-1. Resistin was indirectly positively related to atheromatous MMP activity by way of sVCAM-1. In addition, visceral fat amount was indirectly positively associated with atheromatous MMP activity, by way of adiponectin reduction and resistin elevation.

Conclusion:
Diet and exercise affects atheromatous MMP activity by modulating the systemic inflammatory milieu, with sVCAM-1, resistin, and adiponectin closely interacting with each other and with visceral fat.
Abstract 12: The Effect of Extracellular Matrix Stiffness on Human Bone Marrow-Derived Mesenchymal Stem Cell Differentiation
Authors: Müller Alison L , Li Yun , Hinz Boris , Freed Darren H
Published: July 2021

Differentiation of human mesenchymal stem cells (hMSCs) has been shown to be influenced by the surrounding microenvironment. It is important to understand the physiological implications of the hMSC microenvironment regarding differentiation within the body, especially in patients with cardiovascular disease. We are interested in understanding the influence of differing extracellular matrix (ECM) stiffness found in the body that the MSC encounters during its journey from the bone marrow to the infarct scar in patients recovering from a myocardial infarction. As hMSCs must respond rapidly to their environment, we also investigated the influence of microRNA at different surface tensions.

HMSCs were isolated from the bone marrow of patients undergoing open heart surgery and cultured in standard DMEM/F12 with 20% FBS. We plated these cells on fibronectin-coated plates with surface tensions of 2kPa, simulating bone marrow; 15kPa, simulating left ventricle; and 100kPa, simulating a fibrotic environment. Protein and mRNA were collected for further analysis.

Our data have revealed that softer surface tensions, representing a bone marrow-undifferentiating environment, cause a decrease in the protein expression of EDA-fibronectin and alpha-smooth muscle actin. There were also increases in mRNA of myosin heavy chain-9 and 10, and collagen-1. Softer surface tensions also show a slight increase in miR-301a, although at 15kPa, miR-301a expression is increased even further. Our lab has previously shown that miR-301a is involved in maintaining a proliferative phenotype of hMSCs. Interestingly, Dicer1, responsible for processing microRNAs, is upregulated at softer surface tensions and attenuated at 15kPa. Dicer1 mRNA expression is attenuated at 15kPa. These results indicate that ECM stiffness influences hMSC differentiation and the increase in Dicer1 found with softer matrices could represent an umbrella miRNA inhibition effect to effectively suppress hMSC differentiation.
Abstract 127: Collagen Nanopatterning Modulates Endothelial Cell Atheroprotective Function, Survival, and Angiogenesis
Authors: Huang Ngan F , Lai Edwina , Zaitseva Tatiana , Paukshto Michael V , Cooke John P
Published: July 2021

Vascular endothelial cells (ECs) are longitudinally oriented in regions of laminar flow, but randomly distributed in regions of disturbed flow. The unaligned ECs in disturbed flow fields manifest altered function and reduced survival that promote atherosclerotic lesion formation. We hypothesized that the alignment of the ECs may directly influence their biology, independent of fluid flow. We fabricated parallel-aligned nanofibrillar collagen scaffolds with that mimic the native structure of collagen extracellular matrix within blood vessels, and examined the effects of aligned collagen nanopatterning on EC alignment, function, and in vivo survival. The 30-nm diameter aligned nanofibrils reoriented F-actin assembly along the nanofibril direction. ECs cultured on aligned nanofibrils were also 50% less adhesive for monocytes than the ECs grown on randomly oriented fibrils. To test the efficacy of the aligned nanofibrillar scaffolds in improving neovascularization in vivo, we induced unilateral hindlimb ischemia in SCID mice by excising the superficial femoral artery. The mice received one of the following treatments at the site of the excised femoral artery: 1) aligned nanofibrillar scaffold seeded with human ECs; 2) non-patterned scaffold seeded with ECs; 3) EC delivery in saline; or 4) no treatment (n>4). After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion in the group treated with cell-seeded aligned nanofibrillar scaffolds, in comparison to the groups with no treatment or EC delivery in saline. Furthermore, based on non-invasive bioluminescence imaging, the transplanted ECs persisted for longer periods of time when cultured on aligned nanofibrillar scaffolds, in comparison to non-patterned scaffolds. Together, these studies demonstrate that 30-nm aligned nanofibrillar collagen scaffolds guide cellular organization, modulate endothelial inflammatory response, and enhance cell survival and angiogenesis after implantation in the ischemic hind limb. These results have important implications for therapeutic cell delivery approaches.
Abstract 13: Nkx2-5-notch Signaling Axis Regulates The Proliferation Of The Atrial Myocytes And Conduction System
Authors: Nakano Austin , Nakashima Yasuhiro , Yanez Diana A , Touma Marlin , Nakano Haruko , Jarodzewicz Artur , Jordan Maria C , Pellegrini Matteo , Roos Kenneth P , Roos Kenneth P
Published: July 2021

Rationale:
Tight control of cardiomyocyte proliferation is essential for the formation of four-chambered heart. Although human mutation of NKX2-5 is linked to septal defects and atrioventricular conduction abnormalities, early lethality and hemodynamic alteration in the mutant models have caused controversy as to whether Nkx2-5 regulates cardiomyocyte proliferation.

Objective:
In this study, we circumvented these limitations by atrial-restricted deletion of Nkx2-5.

Method and Results:
Atrial-specific Nkx2-5 mutants died shortly after birth with hyperplastic working myocytes and conduction system including two nodes and internodal tracts. Multicolor reporter analysis revealed that Nkx2-5-null cardiomyocytes displayed clonal proliferative activity throughout the atria, indicating the suppressive role of Nkx2-5 in the cardiomyocyte proliferation after chamber ballooning stages. Transcriptome analysis revealed that aberrant activation of Notch signaling underlies hyperproliferation of mutant cardiomyocytes, and forced activation of Notch signaling recapitulates hyperproliferation of working myocytes but not conduction system.

Conclusion:
Collectively, these data suggest that Nkx2-5 regulates proliferation of atrial working and conduction myocardium in coordination with Notch pathway.
Abstract 135: Reprogramming of Pig Cardiac Fibroblasts to Cardiomyocyte Fate: Implications for Gene Therapy to Treat Myocardial Infarction
Authors: Singh Vivek P , Mathison Megumi , Xu Xueping , Patel Vivek K , Belaguli Narasimhaswamy S , Gibson Brian W , Cooney Austin J , Rosengart Todd K
Published: July 2021

There is an urgent clinical need to develop new therapeutic approaches to treat heart failure, but the biology of cardiovascular regeneration is complex. A new generation of reprogramming technology involves trans-differentiating one adult somatic cell type directly into another. We reported previously that administration of gene transfer vectors encoding Gata4 (G), Mef 2c (M) and Tbx5 (T), reprograms rat cardiac fibroblasts into induced cardiomyocytes (iCMs) in vitro and improves cardiac function in myocardial infarction models. Previous cardiac reprogramming studies were restricted to rodent and human fibroblasts. However studies in large animal models relevant for pre-clinical studies is lacking.

The Aim of the present study was to determine the optimal combination of factors necessary for direct reprogramming of porcine fibroblasts towards a cardiomyocyte lineage for the first time. Here we have used human lentiviral system to express various cardiomyocyte enriched transcriptional regulators such as GMT, Hand2, myocardin, and two microRNAs, miR-590 and mir-199, in porcine cardiac fibroblasts. Reprogramming of fibroblasts into iCMs was determined 4 weeks post-virus transduction by FACS analysis for the activation of endogenous cardiac troponin T (cTnT).

GMT alone was not sufficient to reprogram porcine fibroblasts although this combination was shown previously to be sufficient to convert rodent fibroblasts into iCMs. Addition of mir-199 to GMT was also not sufficient to promote reprogramming. However, adding miR-590 or Hand2, and myocardin (HM) to GMT resulted in reprogramming, although the efficiency remained low. Together, our results show that combination of 5 distinct transcription regulators (GMTHM) or GMT plus mir-590 is necessary for reprogramming porcine fibroblast.

These findings demonstrate that pig fibroblasts can be directly reprogrammed toward the cardiomyocyte lineage, and represent a step toward possible therapeutic application of this reprogramming approach in a pre-clinical setting.
Abstract 137: Pharmacologically Controlled IGF-1 Release For Paracrine Support Of Engineered Heart Muscle
Authors: SOONG POH LOONG , Tiburcy Malte , Christoph Jan , Luther Stefan , Jebran Fawad , Schoendube Fritz , Zimmermann Wolfram H
Published: July 2021

Tissue engineered heart repair is developing rapidly, but needs refinement before clinical translation. We tested the hypothesis that force generating human engineered heart muscle (EHM) can be enhanced by integration of insulin-like growth factor-1 (IGF-1) secreting fibroblasts.

Methods:
TetOn lentiviral particles encoding for IGF-1 and the Tet-transactivator (tTA) were cloned and used to stably transduce human foreskin fibroblasts (HFF). Baseline and doxycycline induced IGF-1 release from HFFTetOn+IGF1 was quantified by ELISA. HFFwt and HFFTetOn+IGF1 conditioned medium was layered over human embryonic stem cell (HES2) derived cardiomyocytes followed by an analysis of AKT-phosphorylation. EHMs were assembled from HES2-derived cardiomyocytes and HFF (HFFwt or HFFTetOn+IGF1) at 70:30 ratio. Transgene activation was induced by addition of doxycycline (10 ng/ml) for 7 days. Twitch forces and response to pharmacological stimuli were measured to assess the functional consequences of IGF-1 release. EHMs were subsequently subjected to morphological analysis or dissociated into single cells to assess cellular composition of EHMs.

Results:
HFFTetOn+IGF1 released IGF-1 upon doxycycline stimulation (3.3x10-6 vs 8.3x10-8 [HFFwt] ng/ml/cell/day. Secreted IGF-1 from HFFTetOn+IGF1 induced Akt phosphorylation in HES2-derived cardiomyocytes (2.4±0.6 fold increase of [HFFwt]; n=3). EHMs with HFFTetOn+IGF1 developed significantly higher twitch forces than EHMs with HFFwt (0.24±0.03 vs 0.15±0.02 mN; n=10) under baseline conditions (IGF-1 leak). Doxycycline induced IGF-1 release further enhanced (P<0.05) EHM twitch force (0.26±0.03 mN; n=10). Single cell analysis from EHMs demonstrated cardiomyocyte hypertrophy in response to paracrine IGF-1 release (154±11% of [HFFwt] control; n=4). Histological analyses demonstrated that HFFTetOn+IGF1 supplemented EHMs contained thicker muscle bundles and enlarged cardiomyocytes.

Conclusion:
EHM can be functionally enhanced by integration of drug-controllable IGF-1 release. Drug controllable, cell based paracrine release of protective factors may not only be exploited to enhance tissue engineered myocardium in vitro but also to achieve better survival and integration of EHM grafts in vivo.
Abstract 140: Effect of Transendocardial Autologous Cardiac Stem Cells and Bone Marrow Mesenchymal Stem Cells to Reduce Infarct Size and Restore Cardiac Function in a Heart Failure Swine Model
Authors: Suncion Viky Y , Karantalis Vasileios , Bagno Luiza L , McCall Frederic , Wang Bo , Golpanian Samuel , Rodriguez Jose E , Rosado Marcos , Valdes David , Gomes Samirah , Gomes Samirah , Da Silva Jose , Premer Courtney , Morales Azorides , Morales Azorides , Balkan Wayne , Mushtaq Muzammil , Zambrano Juan Pablo , Heldman Alan W , Hare Joshua M
Published: July 2021

Background:
A cell combination of human mesenchymal stem cells (MSCs) and c-kit+ cardiac stem cells (CSCs) improves left ventricular (LV) performance to a greater degree than MSCs alone in post myocardial infarction swine. To advance the development of cell combination therapy, we administered autologous swine cells, and tested the hypothesis that transendocardial autologous CSCs/MSCs produces greater improvement of performance than MSCs in a rigorous model of heart failure due to post infarct LV remodeling.

Methods:
Gottingen mini-swine (n=28) underwent LAD coronary artery occlusion followed by reperfusion, and allowed to undergo LV remodeling for 90 days. Autologous MSCs were amplified from bone marrow and CSCs from right ventricular biopsies in each swine, and injections of either CSC/MSC combo (1M/200M, n=7), MSCs (200M, n=7), or placebo (Plasmalyte, n=6) were injected to the infarct-border zone via the NOGA system. Cardiac MRI and pressure volume loops were obtained before and after therapy.

Results:
Both cell groups had substantially reduced scar size (Combo –37.2.9± 5.4% vs MSCs –38.8±7.5% vs placebo −7.2±6.3, P=0.0001) and increased viable tissue (Combo +30.9±7% vs MSCs +41.8±10.5% vs placebo +7.7±4.5, P<0.0001) relative to placebo. Ejection Fraction (EF) improved only in the Combo group (Combo +7.0±2.8 vs MSCs +3.4±1.3 vs placebo +1.2±1.6 EF units, P=0.04). Accompanying this EF restoration was a substantial improvement in the Combo group in stroke volume (Combo +47.2±11.1% vs MSCs +32.6±12.0% vs placebo +10.8±4.5, P<0.0001), cardiac output (Combo +35.9±7.6% vs MSCs 41.9±26.5% vs placebo −16.4±6.6%, P=0.01) and diastolic strain rate (Combo +18.9±8.6% vs MSCs 14.0±8.8% vs placebo −14.9±9.5%, P=0.03).

Conclusions:
Combination cell therapy and MSCs alone dramatically reduce scar size in a swine model of chronic ischemic cardiomyopathy. In contrast, combination therapy has much greater impact on functional recovery, increasing EF to [near normal] levels. These findings illustrate that interactions between ckit+ CSCs and MSCs result in substantial enhancement in cardiac performance, establish the safety of autologous cell combination strategies, and support the development of an advanced second generation cell therapeutic product.
Abstract 146: Biphasic Effect Of Cyclic Amp Axis On Cardiomyocyte Survival
Authors: Aoyama Morihiko , Bando Yasuko K , Kawase Haruya , Monji Akio , Murohara Toyoaki
Published: July 2021

Background:
Persistent cardiac hypertrophy in response to pathological stimuli is results in maladaptive myocardial remodeling and cell death. Clinical evidence revealed that one of the most significantly beneficial medications for targeting heart failure with pathologic myocardial hypertrophy is beta1-adrenergic receptor (β1R) blockers. The molecular pathway of β1R is mediated by the second messenger cyclic AMP (cAMP). However, there are some debate regarding the role of cAMP in myocardial survival. We hypothesized whether there may be threshold concentration of cAMP in cell susceptibility to cardiomyocyte cell death.

Methods:
Male 14-week-old C57BL6 mice were subjected to the surgery of thoracic aortic constriction (TAC) to induce pressure overload. Changes in apoptosis were evaluated in each heart section and in vitro culture of neonatal cardiomyocytes using TUNEL. To elucidate the concentration-dependent distinct effect of cAMP on myocardial cell death, we tested the different concentration of cell-permeable cAMP (8-br-cAMP) at low (60 μM) and high concentration (6 mM), and receptor-mediated cAMP-stimulators (Ex4; exendin-4, ISO; isoproterenol).

Results:
In vitro analysis revealed that the high-cAMP and ISO exhibited marked increase in TUNEL-positivity (15.46%±3.09% for high-cAMP versus 6.71%±0.33% for ISO), which was reversed by Rp-cAMP (1.80%±0.17% and 2.05%±0.25%, respectively). Unexpectedly, the 8-p-Methoxyphenylthon-2-O-methyl-cAMP (pMe-cAMP, 50 μM), the specific activator of another cAMP-sensitive target Epac, reversed the high-cAMP-induced cell death even at a less extent compared to that observed by PKA-inhibitor Rp-cAMP (3.73%±0.70%). Serum depletion induced 3.22±0.24% of TUNEL-positive cell count of NRVM, which was reversed by pMe-cAMP, (50 μM) and Ex4 (1.74±0.18%, n=6, P<0.01), which was insensitive to PKA inhibition by Rp-cAMP (100 μM).

TAC increased myocardial apoptosis. TAC-CON heart exhibited 1.66-fold decrease in cardiac cAMP concentration compared to sham-CON. Ex4 ameliorated the TAC-induced cardiac dysfunction and apoptosis by increase in cAMP.

Conclusions:
The cAMP-related cell death was mediated by PKA activation, which were reversed by Epac activation.
Abstract 15: Limiting GRK2 in the Ischemic Heart can Promote Cardiac Regeneration
Authors: Scimia Maria Cecilia , Zuo Lin , Sydnes Kate E , Zuppo Daniel A , Gao Erhe , Koch Walter J
Published: July 2021

The detrimental role of G protein-coupled receptor (GPCR) kinase (GRK2) following cardiac injury/stress has been documented over the last two decades. Importantly, our lab has shown that inhibition or deletion of GRK2 in cardiomyocytes can prevent and also rescue heart failure (HF) phenotypes. Its role in GPCR desensitization including regulation of β-adrenergic receptors (βARs) during HF development has been well characterized. However, recently our lab and others have found that GRK2 can have novel GPCR-independent effects in the heart that appear to contribute to its pathological effects and thus, inhibition of these actions of GRK2 may contribute to therapeutic effects seen. In this study we explored whether the cardiac repair observed with lower myocardial GRK2 might involve regenerative processes. In cardiac-specific GRK2 knockout (KO) mice and also transgenic mice with cardiac-targeted expression of the βARKct, a peptide inhibitor of GRK2 activation via Gβγ sequestration, we induced HF via coronary artery ligation and subsequent myocardial infarction (MI) and measured aspects of cardiac repair including potential regeneration indices. Post-MI mice (GRK2 KO, βARKct mice and wild-type and non-transgenic control mice) were treated with 5-ethynyl-2’-deoxyuridine (EdU) or Bromodeoxyuridine (BrDU) to examine indices of DNA proliferation in myocytes as well as Ki67 staining. We also quantitated c-kit+ cells and myocytes in the post-MI hearts to compare how either loss of GRK2 expression or inhibition via its C-terminus altered potential regeneration mechanisms compared to control mice with endogenous GRK2 levels and activity. We found significantly more BrDU positive myocytes in post-MI hearts with lower GRK2 and this correlated with increased myocytes that were also cKit+. Thus, it appears that the myocardial functional improvement seen in the post-MI heart with targeted lowering of GRK2 involves, to at least a certain extent, regenerative mechanisms. This adds novel insight into the therapeutic potential of GRK2 inhibition for HF.
Abstract 157: Toll like Receptor 4 Activation Promotes Cardiac Arrhythmias By Decreasing The Transient Outward Potassium Current (ito) Through An Irf3 dependent And Myd88 independent Pathway
Authors: Medei Emiliano , Monnerat-Cahli Gustavo , Alonso Hiart , Gallego Monica , Lopez Alarcon Micaela , Bassani Rosana A , Casis Oscar
Published: July 2021

Cardiac arrhythmias are one of the main causes of death worldwide. Several studies have shown that inflammation plays a key role in different cardiac diseases and Toll like receptors (TLR’s) play an important role in cardiac complications. In the present study, we investigated whether the activation of TLR4 induces cardiac electrical remodeling and arrhythmias. Also the signaling pathway involved in these phenomena was studied. Action potentials, the presence of cardiac arrhythmias and transient outward K
+
current (I
to
) were recorded in Wistar rat’s hearts after 24 h exposure to the TLR4 agonist ultrapure Lipopolysaccharide (LPS - 1μg/ml). TLR4 stimulation
in vitro
promotes a cardiac electrical remodeling that leads to cardiac action potential prolongation which evokes arrhythmic events such as delayed after depolarization (DAD's) and triggered activity. The perfusion of LPS (1μg/ml) during 30 minutes did not modify I
to
. Conversely, after 24 h of LPS incubation I
to
was reduced, with no changes in the biophysical properties of the current. Major changes in Ca
2+
cycling were not observed in ventricular myocytes after 24 h exposure to LPS; however, extrasystolic activity was present in a considerable number of cells (25%). Neither the blockade of Interleulink-1 receptor-associated kinase 4 nor nuclear factor kappa B (NF-kB) prevented the LPS effect on I
to
. However, interferon regulatory factor 3 (IRF3) inhibition prevented the effect of TLR4 activation on I
to
. Activation of TLR4 induced extrasystolic activity, longer AP duration and evoked DAD's and triggered activity because of a reduction in I
to
. The mechanism involved is MyD88-independent and IRF3-dependent.
Abstract 158: Ultrasound-Induced Inhibition and Modulation of Neonatal Ventricular Cardiomyocyte Depolarization
Authors: Mehta Natasha , Kresh J Yasha , Kutalek Steven P , Lewin Peter A , Kohut Andrew R
Published: July 2021

Background:
Ultrasound can interact with tissue through either thermal or non-thermal physical mechanisms. Radiation force has been shown to stimulate cardiac and neural tissue in vivo. Ultrasound might hold clinical potential as a noninvasive therapeutic tool via specific bioeffects on cardiomyocytes. This study aims to assess the effect of ultrasound on cardiomyocyte depolarization in a tissue culture model.

Methods:
Cardiomyocytes were isolated from neonatal rat ventricular tissue and plated directly on microelectrode arrays to record depolarization patterns. A custom 2.5 MHz unfocused ultrasound transducer was directed at the cardiomyocytes in a tissue culture model. A function generator, with an amplified signal +50 dB, delivered acoustic energy at variable settings of 0.1, 0.3, 0.5 and 1.0 Vpp, pulse durations of 2, 5 and 10 ms, and burst periods of 100, 250 and 300 ms. Five trials were conducted at each setting (36 total trials) with 30s of continuous ultrasound exposure followed by an off interval of 1 minute.

Results:
The R-R interval durations (ID) were measured throughout the recording period. Prior to ultrasound delivery, the IDs were highly irregular, ID range = 0.3-2.7 s. As ultrasound was delivered in an asynchronous manner, using 0.1 and 0.3 Vpp and PD = 2 and 5 ms, there was suppression/inhibition of cellular depolarization for the first 5-10 s. Then 10-15 s after the start of ultrasound delivery, the depolarization rate increased and demonstrated less R-R interval variability (ID=0.88-1.03 s, P value<0.05), even after the ultrasound exposure.

Conclusion:
Ultrasound can inhibit and modify the frequency of spontaneous electrical depolarizations of neonatal ventricular cardiomyocytes in a tissue culture model. Our observations could be due to conditioning via stretch and compression-mediated mechanosensitive pathways, by modifying intracellular calcium handling or altering cell signaling.
Abstract 159: Ablation of the Hypertension Candidate Gene
ATP2B1
Leads To Deficient Calcium Cycling, Systolic Dysfunction and Heart Failure Following Pressure Overload
Authors: Stafford Nicholas P , Zi Min , Neyses Ludwig , Cartwright Elizabeth J
Published: July 2021

Mutations in
ATP2B1
encoding the ubiquitous calcium extrusion pump Plasma Membrane Calcium ATPase 1 (PMCA1) have recently identified it as having the strongest association of any gene to hypertension, yet the role of PMCA1 in the pressure-overloaded heart is not known. To investigate this we generated a novel mouse line carrying cardiomyocyte-specific deletion of PMCA1 (PMCA1
cko
) and challenged them with transverse aortic constriction (TAC) alongside littermate ‘floxed’ controls (PMCA1
f/f
). After two weeks, echocardiographic analysis revealed signs of systolic dysfunction and left ventricular (LV) dilation in PMCA1
cko
hearts as evidenced by reduced fractional shortening and increased diastolic diameter (both p<0.05), whilst function in PMCA1
f/f
TAC controls remained preserved. This was accompanied by an increase in normalised lung weight in PMCA1
cko
mice compared to sham operated and TAC controls (p<0.05) indicative of pulmonary congestion and a progression into LV failure, despite comparable hypertrophic growth amongst the two TAC cohorts. Hemodynamic analysis following LV catheterisation revealed contractility, as measured by left ventricular elastance (E
es
), to be increased in controls after TAC (PMCA1
f/f
TAC 12.69 ± 1.63 vs sham 7.02 ± 1.11 mmHg/μl, p<0.05), a change which was not reciprocated in knockout hearts (PMCA1
cko
TAC 7.70 ± 1.19 vs sham 7.22 ± 1.55 mmHg/μl). To examine whether altered calcium handling could be the underlying cause of the observed phenotype, cardiomyocytes were isolated following one week TAC and loaded with Indo-1, prior to the onset of failure in PMCA1
cko
hearts. Compatible with an increase in E
es
, systolic calcium levels were higher in PMCA1
f/f
myocytes following pressure overload compared to sham controls (p<0.05), whilst PMCA1
cko
TAC myocytes displayed equivalent peak calcium levels to their respective sham controls. These results suggest that PMCA1 may play a necessary role in enhancing calcium cycling during the early response to pressure overload, and that disrupting this gene may increase the susceptibility to heart failure under these conditions. This may provide first evidence of a novel genetic basis for the development of heart failure in a proportion of hypertensive patients.
Abstract 166: Matrix Metalloproteinase-9 Mediates Post-hypoxic Vascular Pruning Of Cerebral Blood Vessels By Degrading Laminin And Claudin-5
Authors: Boroujerdi Amin , Welser-Alves Jennifer V , Milner Richard
Published: July 2021

Objective:
Vascular remodeling involves a highly coordinated break-down and build-up of the vascular basal lamina and inter-endothelial tight junction proteins. The goal of this study was to examine the role of matrix metalloproteinase-9 (MMP-9) in remodeling of cerebral blood vessels, both in hypoxia-induced angiogenesis and in the vascular pruning that accompanies the switch from hypoxia back to normoxia.

Approach and Results:
In a chronic mild hypoxia model of cerebrovascular remodeling, gel zymography revealed that MMP-9 levels were increased, both in the hypoxic angiogenic response and in the post-hypoxic pruning response. Compared to wild-type mice, MMP-9 KO mice showed no alteration in hypoxic-induced angiogenesis, but did show marked delay in post-hypoxic vascular pruning. In wild-type mice, vascular pruning was associated with fragmentation of vascular laminin and the tight junction protein claudin-5, while this process was markedly attenuated in MMP-9 KO mice. In vitro experiments showed that hypoxia stimulated MMP-9 expression in brain endothelial cells (BECs) but not pericytes. While immunofluorescent and flow cytometry analyses showed that hypoxia led to reduced expression of laminin and claudin-5 in wild-type BECs, this decrease was absent in MMP-9 KO BECs.

Conclusions:
These results show that while MMP-9 is not essential for hypoxic-induced cerebral angiogenesis, it plays an important role in post-hypoxic vascular pruning by degrading laminin and claudin-5. Our data support the concept that MMP-9 inhibition might provide therapeutic benefit in the treatment of ischemic stroke, by preventing post-hypoxic vascular pruning, thereby optimizing vascular density and integrity.
Abstract 167: Growth Induction to Reverse Congenital Aortic Developmental Hypoplasia
Authors: Labuz Daniel F , Berry James M , Pyles Lee A , Foker John E
Published: July 2021

Objectives:
Congenital heart disease may include hypoplastic heart valves, ventricles or great arteries. Infants with coarctation of the aorta (CoA) often have a hypoplastic transverse aortic arch (TAA) which can greatly complicate surgical repairs. Although these defects are often considered to be genetic in origin, our hypothesis was that they are problems of development which are potentially reversible. We tested the corollary hypothesis that hypoplastic TAAs retain normal developmental potential and the increased aortic flow after CoA repair provides the biomechanical signal for catch-up growth.

Methods:
Infants (N = 19) with TAA hypoplasia who underwent surgical CoA repair were studied for TAA growth by echocardiography done prior to and at intervals up to 13 years later. The TAA diameters were indexed using nomograms and calculated as standard deviations from expected size (SDE). Normal range is ± 2 SDE and < −2 indicates hypoplasia.

Results:
1) TAA growth was rapid and significant within 3 months. 2) The initial average TAA SDE = −3.7 (range: −7.6 to −2.1) (0/19 normal); 3 months = −1.5 (-4.1 to 0.9) (12/17); 1 year = −1.0 (-4.2 to 1.3) (15/19); 5 to 13 years = −0.6 (-2.1 to 0.9) (16/17 normal).

Conclusions:
1) Hypoplastic aortic arches grew rapidly to normal size with increased flow following CoA repair. 2) The results suggest the cause of hypoplasia was underdevelopment from low flow and, when increased, flow provided the biomechanical signal to induce catch up growth. 3) Infants were a relevant model for demonstrating the aortic growth signal. 4) Growth induction by increased blood flow could be used to reverse aortic underdevelopment in other selected patients.