bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 52: Temporal Regulation Of Cardiac Cytokine Expression in Response to Chronic β-Adrenergic Receptor Stimulation
Authors: Grisanti Laurel A , Repas Ashley A , Carter Rhonda L , Talarico Jennifer A , Gold Jessica L , Koch Walter J , Tilley Douglas G
Published: July 2021

Chronic catecholamine stimulation of β-adrenergic receptors (βAR) is ultimately deleterious during heart failure (HF). While alterations in cytokines contribute to HF pathogenesis and βAR have been demonstrated to regulate cytokines in different models of HF, a comprehensive understanding of this relationship is lacking. Thus, we sought to characterize the impact of chronic βAR signaling on cardiac cytokine expression in vivo. C57BL/6 mice underwent infusion with vehicle or isoproterenol (Iso; 3 mg/kg/day) via minipumps for 1 or 2 weeks and cardiac function was monitored via echocardiography. At study termination, hearts were excised and assessed for changes in hypertrophy, fibrosis and apoptosis, each of which were enhanced by Iso. Expression of cardiac transcripts were assessed via whole transcriptome analysis, where 780 and 689 transcripts were significantly altered at 1 and 2 weeks of Iso, respectively, with only 115 transcripts regulated similarly between the two cohorts. Significant changes in cytokine transcript expression was observed in response to chronic Iso and Ingenuity Pathway Analysis (IPA) predicted the involvement of additional upstream cytokine regulators potentially regulated by Iso. Transcriptome results and IPA predictions were confirmed via qRT-PCR. A cytokine array also confirmed temporally-distinct alterations in the expression of 42 cytokines at the protein level. Differential alterations in cytokine expression resulting from 1 versus 2 weeks of Iso infusion suggest that cytokine-directed therapies may have distinct temporally-dependent consequences on cardiac function and survival under conditions of chronic catecholamine stress.
Abstract 55: Opposing Roles Of Distinct Macrophage Lineages In Heart Failure And Cardiac Recovery
Authors: Lavine Kory J , Epelman Slava , Uchida Keita , Weber Kassandra J , Schilling Joel D , Nichols Colin G , Randolph Gwendalyn J , Mann Douglas L
Published: July 2021

Why inflammation is simultaneously deleterious following injury and essential for tissue repair continues to be fundamentally important and debated question. Recently, a new paradigm has emerged in the macrophage field: that organs are replete with resident macrophages of embryonic origin, distinct from monocyte-derived macrophages. This added complexity raises the question of whether distinct immune cells drive inflammatory and reparative activities following injury. Previous work has demonstrated that the neonatal heart has a remarkable capacity for tissue repair compared to the adult, offering an ideal context to examine these concepts. We hypothesized that unrecognized differences in macrophage composition in the neonatal and adult heart represents a key determinant of cardiac recovery. To test this hypothesis, we generated a novel cardiomyocyte ablation model and demonstrated that following injury neonatal mice expand a population of resident cardiac macrophages derived from embryonic lineages, which generate minimal inflammation and are necessary and sufficient for cardiac recovery through promotion of cardiomyocyte proliferation and angiogenesis. During homeostasis the adult heart also contained embryonic-derived macrophages with similar properties. However, following injury these cells disappeared, and instead, the adult heart recruited pro-inflammatory monocytes and monocyte-derived macrophages that lacked reparative activities. Inhibition of monocyte recruitment into the injured adult heart preserved embryonic-derived macrophage subsets, reduced inflammatory cytokine and chemokine production, and enhanced tissue repair. Together, these findings indicate that embryonic-derived macrophages, rather than monocyte-derived macrophages, are key mediators of cardiac recovery. Therapeutics targeting distinct macrophage and monocyte lineages may serve as novel treatments for heart failure.
Abstract 6: Formation of Human Heart Muscle Directly from Embryonic and Induced Pluripotent Stem Cells
Authors: Hudson James E , Tiburcy Malte , Zimmermann Wolfram-Hubertus
Published: July 2021

Tissue engineering enables the simulation of human heart physiology and pathology. It typically requires a mixture of cardiomyocytes, stromal cells, and extracellular matrix for fabrication. Here, we hypothesised that bioengineered heart muscle (BHM) can be formed directly from undifferentiated pluripotent stem cells by triggering processes of embryonic cardiogenesis.

Methods and Results:
We tested our hypothesis by applying an optimized serum-free cardiac differentiation protocol to undifferentiated pluripotent stem cells in a collagen type 1 hydrogel. During this process BHMs traversed through distinct developmental stages: early mesoderm (3 days), cardiac specification (10 days), and cardiac maturation (up to 50 days). Flow cytometry demonstrated that BHMs are comprised of primarily cardiomyocytes (α-actinin positive cells, 51 ± 5%, n = 6) and stromal cells (CD90 positive cells, 41 ± 5%, n = 6), with low yields of contaminating cells. By 22 days the BHMs exhibited measurable contractile force (207 ± 19 μN) and contained elongated cross-striated cardiomyocytes.

We next sought to optimize the force of contraction and also the maturity of the BHM, by investigating the effect of mechanical stimuli and growth factors. Mechanical stimulation was essential for BHM formation. Additionally, we found that 2 developmentally important growth factors, FGF2 and TGFβ1, induced pathological and physiological hypertrophy, respectively. This was characterized by an increase in cell size in both conditions coupled with reduced force and higher ANP expression in FGF2 treated BHM, and a higher force with reduced ANP expression and elevated β-MHC/α-MHC ratio TGFβ1 treated BHM.

Using our optimized protocol, 28 day old BHM responded to electrical pacing, preloading, and inotropic stimuli similarly as bona fide myocardium.

Conclusion:
BHM can be formed directly with undifferentiated pluripotent stem cells by recapitulating normal cardiac development. The serum-free protocol with developmentally defined stimuli provides us with a useful in vitro model to study cardiac biology and potentially provides a method of producing cardiac tissue for regenerative applications.
Abstract 65: Regulation of Cardiac Hypertrophy and Dilated Cardiomyopathy by CIP
Authors: Huang Zhan-Peng , Kataoka Masaharu , Chen Jinghai , Wang Da-Zhi
Published: July 2021

Cardiac hypertrophy is one of the primary responses of the heart to pathophysiological stress. However, the mechanism of the transition from compensative hypertrophic growth to cardiac dilation is poor understood. Recently, we identified a cardiac-specific expressed gene CIP. The expression of CIP is unchanged in hypertrophic heart but significantly down-regulated in dilated hearts, suggesting CIP may play an important role in the transition from cardiac hypertrophy to dilated cardiomyopathy.

We generated CIP knockout mice and found that CIP is dispensable for cardiac development. Interestingly, CIP-null mutant mice developed severe cardiac dilation 4 weeks after TAC (transverse aortic constriction) surgery, while control mice were still at the stage of compensative hypertrophic growth. Echocardiography and histological examinations showed that mutant hearts had enlarged chamber with thinner ventricle wall and decreased cardiac performance compared to controls. The expression of marker genes of cardiac disease, BNP and Myh7, was elevated. Consistently, deletion of CIP in Myh6-CnA transgenic mice result in premature death, displaying severe left ventricle dilation.

Conversely, cardiac-specific CIP overexpression inhibited pressure overload-induced cardiac hypertrophy. CIP transgenic mice exhibit decreased ventricle weight/body weight ratio, decreased cardiomyocyte cross-section area and repressed expression of hypertrophic related marker genes. CIP overexpression also protected the heart from developing cardiac dilation and preserved the cardiac function after prolonged pressure overload.

We performed unbiased microarray assay to document the transcriptome in CIP knockout and control mice which were subjected to pressure overload (TAC). The analysis of Gene Ontology term indicated the Negative Regulation of Apoptosis was down-regulated while the Collagen/Extracellular Structure Organization was up-regulated in CIP-null hearts under TAC condition.

In summary, our studies established CIP as a key regulator of the transition from cardiac hypertrophy to dilated cardiomyopathy. The protective effect of CIP in cardiac remodeling indicates that CIP could become a therapeutic target for cardiac diseases.
Abstract 66: Unique Cardiac Fibroblast Expression Profiles in Pathological and Physiological Hypertrophy
Authors: Lighthouse Janet K , Velasquez Lissette S , Small Eric M
Published: July 2021

The heart undergoes hypertrophic growth in response to both physiological and pathological stimuli. Pathological hypertrophy results from various humoral, mechanical, or ischemic insults, and often leads to cardiac fibrosis, diminished contractility, and heart failure. In contrast, physiological hypertrophy is an adaptive response to the excessive demands of exercise or pregnancy and does not lead to fibrosis. We hypothesized that cardiac fibroblasts, the main cellular source of extracellular matrix in the heart, exhibit distinct expression profiles in physiological or pathological remodeling that influence the divergent fibrotic response. To investigate these differences, we obtained the expression profile of cardiac fibroblasts isolated from mice subjected to swim training, pressure-overload induced cardiac remodeling, or myocardial infarction by RNA-sequencing. Although we observed cardiac growth in all conditions, pressure-overload induced hypertrophy and myocardial infarction induced the predicted fibrotic gene expression signature, which was absent in physiological hypertrophy. Utilizing these validated datasets, we identified novel genes and molecular pathways that are differentially expressed in physiological and pathological hypertrophic remodeling and will correlate transcriptional programs with altered gene profiles. Fibroblast gene expression profiles in pathological and physiological are expected to lead to diagnostic or prognostic markers of fibrotic remodeling as well as genes that may serve as novel therapeutic strategies to prevent or reverse cardiac fibrosis.
Abstract 68: Caveolae-specific Phosphorylation of L-type Calcium Channel β2a Subunit exaggerates Cardiac Hypertrophic Responses after α1 Adrenergic Stimulation in Mice
Authors: Nakayama Hiroyuki , Kumagai Shohei , Matsunami Sachi , Hayamizu Nao , Tonegawa Kota , Otsuka Wataru , Fujio Yasushi
Published: July 2021

Rationale:
Ca2+ influx via L-type Ca2+ channels (LTCCs) plays a pivotal role in excitation-contraction coupling and cardiac hypertrophy. Phosphorylation of LTCC β2a subunit (β2a) by CaMKII enhances channel activity. LTCCs are localized in both T-tubules and caveolae, but the functional role of phosphorylation of β2a in caveolae remains unelucidated.

Objective:
To develop a novel tool to analyze caveolae specific activation of CaMKII and to determine the functional roles of caveolae-specific CaMKII signaling in LTCC-related cardiac hypertrophy.

Methods and Results:
To evaluate caveolae-specific activation of CaMKII, we generated a fusion protein composed of the cytosolic domain of phospholamban (PLN) as a phosphopeptide tag and caveolin3 (cPLN-Cav3). Activation of CaMKII was assessed by phospho-specific antibody for PLN (Thr17). To inhibit caveolae-specific activation, we generated a GFP fusion protein with caveolae-targeting sequences fused to CaMKII inhibitory peptide (CTS-GFP-AIP). In neonatal rat cardiomyocytes (NRCM), adenoviral expression revealed that CTS-GFP-AIP co-localizes with caveolin3 and mediates caveolae specific inhibition of CaMKII, thus validating this novel method. CTS-GFP-AIP inhibited CaMKII phosphorylation of β2a in NRCM, thus suggesting that phosphorylation of β2a occurs exclusively in caveolae. Phenylephrine stimulation mediates CaMKII activation in caveolae, which leads to CaMKII-specific phosphorylation of β2a in vitro and in vivo. Finally, we generated non-phospho mutant β2a -overexpressing mice and assessed hypertrophic responses in both wild-type and mutant β2a transgenic animals (TG). Protein expression by transgenes in mutant TG was similar to those previously reported in wild-type β2a overexpressing mice. Wild-type β2a TG showed exaggerated cardiac hypertrophic responses at two weeks after phenylephrine stimulation when compared to controls (4.8 ± 0.2 vs. 5.5 ± 0.3 for heart weight to body weight ratio; p<0.05), which was completely abolished in mutant TG.

Conclusions:
We developed a novel method to analyze caveolae-specific activation of CaMKII and confirmed that caveolae-specific phosphorylation of β2a exaggerates cardiac hypertrophy caused by phenylephrine stimulation.
Abstract 70: Domain-Specific Roles for GRK2 in Cardiac Hypertrophy and Heart Failure
Authors: Schumacher Sarah M , Gao Erhe , Gold Jess I , Cohen Maya , Chuprun Kurt , Koch Walter J
Published: July 2021

During heart failure (HF), cardiac levels and activity of the G protein-coupled receptor (GPCR) kinase (GRK) GRK2 are elevated, increasing phosphorylation, desensitization and down-regulation of β-adrenergic receptors (βARs) and other cardiac GPCRs. Increased GRK2 participates in adverse remodeling and contractile dysfunction during HF, while inhibition via a carboxy-terminal peptide, βARKct, enhances heart function and can prevent HF development. Mounting evidence supports the idea of a dynamic “interactome” in which GRK2 can uncouple GPCRs via novel protein-protein interactions. Several novel GRK2 interacting partners are important for adaptive and maladaptive myocyte growth including Gq, the signaling trigger for maladaptive cardiac hypertrophy, leading to HF. Importantly, GRK2 contains a putative amino-terminal Regulator of G protein Signaling (RGS) domain (βARK-RGS). This domain directly interacts with Gq and appears to inhibit signaling without altering Gq enzymatic activity. Therefore, this domain may alter hypertrophic responses in the heart and represent a novel role for GRK2 and a potential therapeutic target to limit maladaptive cardiac hypertrophy. We have begun to address this by generation of novel transgenic (Tg) mice with cardiac-specific expression of the RGS domain of GRK2. Using a trans-aortic constriction (TAC) model of pressure overload hypertrophy, we found that expression of βARK-RGS demonstrates anti-hypertrophic effects. Echocardiographic analysis post-TAC revealed reduced left ventricular posterior wall thickness (LVPW) in βARK-RGS compared to non-transgenic littermate controls (NLC) (0.85 vs 1.0 mm LVPWd at 4 weeks). RT-PCR analysis found decreased hypertrophic factor transcripts, such as ANF for which the nearly 18-fold increase post TAC was completely inhibited in βARK-RGS mice. Further, the progression to HF was inhibited in βARK-RGS mice, but not NLCs, 14 weeks post-TAC. While mechanistic characterization is underway, these data support our hypothesis that the RGS domain of GRK2 may serve as a non-canonical inhibitor of Gq-mediated hypertrophic signaling in the heart and highlight how this research may pave the way for novel GRK2-based therapeutic approaches to prevent hypertrophy and HF.
Abstract 76: Meox1 is a Cell-cycle Oscillator Required for Mitotic Transition and Proliferation in Cardiac Fibroblasts
Authors: Higo Shuichiro , Asano Yoshihiro , Masumura Yuki , Sakata Yasushi , Kitakaze Masafumi , Takashima Seiji
Published: July 2021

Background:
Tissue fibrosis plays important roles in the pathogenesis of chronic diseases, including heart failure. The mechanism underlying interstitial fibroblast proliferation is a promising analytical target for therapeutic applications. Here we developed quantitative epigenome profiling to identify a critical regulator in interstitial cell populations that emerges during the progression of heart failure.

Methods and Results:
We subjected pressure-overloaded hearts of mice to trimethylated histone H3 lysine 4 (H3K4me3) ChIP-sequence and RNA-sequence. Expression analysis followed by quantitative H3K4me3 profiling identified 45 fibrosis-related genes with significant H3K4me3 enrichment in failing hearts, including Meox1 transcription factor. Meox1 emerged in the interstitial fibrotic region in failing heart, and intriguingly Meox1 was expressed in the limited population of cardiac fibroblasts both in vivo and in vitro. Meox1-positive fibroblasts were increased in response to a paracrine signal from cardiomyocytes, and knockdown of Meox1 completely inhibited the reactive proliferation of cardiac fibroblasts stimulated by conditioned medium from cardiomyocytes. Gene expression profiling combined with siRNAs clarified that Meox1 depletion resulted in down regulation in the mitosis-related genes including Aurora B kinase. Indeed, Meox1 depletion decreased the cells under mitosis, but conversely increased the proportion of DNA synthesizing cells, thereby inhibited mitotic transition. The cell-cycle synchronization analysis and promoter analysis using live-cell imaging clarified that Meox1 oscillated throughout the cell-cycle and specifically emerged in G2/M phase. Finally, we revealed that Meox1 heterogenously expressed in the interstitial fibrotic are of human ventricular heart tissues from patients with end-stage heart failure. Notably, Meox1 expression was significantly correlated with the fibrosis-related genes in diseased ventricular heart tissues (n=15), suggesting the pathological relevance in clinical settings.

Conclusion:
Our findings identify a novel cell-cycle regulator and propose that Meox1 is a potential target for therapies aimed at preventing tissue fibrosis.
Abstract 81: Sphingosine 1-phosphate Signaling Contributes To Cardiac Inflammation, Remodeling And Dysfunction Following Myocardial Infarction.
Authors: Zhang Fuyang , Xia Yunlong , Wang Wei , Yan Wenjun , Yang Lu , Li Yan , Zhou Fen , Tao Ling
Published: July 2021

Sphingosine 1-phosphate (S1P) generated by sphingosine kinases (SphK) regulates multiple pathophysiological processes in cardiovascular system. The study was designed to investigate the role of SphK/S1P axis in myocardial infarction (MI) induced heart failure and the underlying mechanisms. Male C57BL/6J mice were subjected to permanent left coronary artery occlusion for 4 weeks. The infarcted heart showed increased SphK1 expression and increased S1P content. S1PR1, the predominant type of S1P receptors expressed in left ventricle, was upregulated by 3-fold in cardiac tissue following MI (n=6, all P<0.05). Further, we observed that S1P significantly activated NF-κB/STAT3 signaling and upregulated pro-inflammatory cytokines (TNF-α and IL-6) expression in a dose-dependent manner in cultured neonatal rat ventricular cardiomyocytes (n=3, all P<0.01), all of which were almost blocked by pretreatment with S1PR1 siRNA or FTY720, a functional S1PR1 inhibitor (n=3, all P<0.01). In vivo, administration of FTY720 (3mg/kg, i.p. daily for 4 weeks) decreased SphK1/S1P/S1PR1 axis, inhibited persistent NF-κB/STAT3 signaling activation and blocked pro-inflammatory cytokines production in post-MI heart. Accordingly, FTY720 remarkably alleviated cardiac fibrosis and dysfunction (ejection fraction: 35.5±3.0% vs. 29.4±2.7% , P<0.05). Intriguingly, administration of SphK2 inhibitor ABC294640 (5mg/kg, i.p. daily for 4 weeks) amplified cardiac SphK1/S1P/S1PR1 axis, augmented NF-κB/STAT3 signaling activation and increased inflammatory cytokines expression. Consequently, SphK2 inhibition reduced survival rate (68.4% vs. 83.3%, P<0.05) and exacerbated cardiac maladaptive structural changes and ventricular dysfunction induced by MI (ejection fraction: 25.4±2.3% vs. 30.4±3.2%, P<0.05). Our results provide the evidence that upregulated SphK1/S1P/S1PR1 axis links persistent NF-κB/STAT3 activation, chronic cardiac inflammation and heart failure progression following MI, whereas SphK2 serves as an endogenous suppressor of pathological S1P signaling. Targeting SphK1/S1P/S1PR1 axis by FTY720 treatment or enhancing SphK2 activity might be a potential therapeutic strategy for ischemic heart failure.
Abstract 82: Regulation Of Frataxin By HIF-1 In The Ischemic Diabetic Heart
Authors: Amin Raj H , AlAsmari Abdullah , Nanayakkari Gayani , Quindry John , Mouli Shavanthi , Eldoumani Haitham , McGinnis Graham , Fu Xiaoyu
Published: July 2021

Background:
Diabetes is at epidemic proportions, with the major form of fatality due to congestive heart failure triggered by myocardial infarction (MI). The impaired insulin signalling in the diabetic heart leads to myocardial energy dysregulation that compromises the cardioprotective mechanism against ischemic injury. Therefore understanding how mitochondrial energetics is altered in the diabetic ischemic heart would greatly advance the knowledge base for improving outcomes from heart failure in diabetic patients.

Methods/Findings:
We observed that db/db mice (leptin deficient, type 2 diabetic mice) have increased infarction size (>30%) compared to wild type mice after ischemia/reperfusion (IR) injury by TTC stain. We also found that activity of Hypoxia inducible factor-1 (HIF1) is involved in the cardioprotective response to ischemia, is impaired in db/db hearts. HIF1 is known to transcriptionally regulate genes involved in myocardial energetics. We recently found that HIF1 transcriptionally regulates the mitochondrial protein frataxin (Fxn) in cardiomyocytes as determined by luciferase assays (>3 fold). In vitro studies indicate that hypoxic conditions increase Fxn protein expression in cardiomyocytes as determined by western analysis (2 fold). Fxn plays an important role in the Fe-S cluster biogenesis required for aconitase, succinate dehydrogenase and complexes in the mitochondria. Interestingly, we observed decreased expression of Fxn in the ischemic diabetic heart.

Conclusion:
we postulate that attenuated HIF1-Fxn signalling in ischemic diabetic heart leads to abnormally enlarged infarction size in response to IR. The decline in HIF-1 activity in response to hypoxia was further validated in cardiomyocytes cultured in high glucose media. The significance for Fxn against hypoxic injury was confirmed by utilizing overexpressed Fxn cardiomyocytes via MTT, ATP and aconitase activity assays. Current and future work: currently we are attempting to identify the HIF response element (HRE) in Fxn promoter to further validate the transcriptional activity of HIF1. In addition, we are completing the IR surgeries on HIF1 KO mice to address the cardioprotective nature of HIF1-Fxn signalling against MI.
Abstract 94: Comparison of Ultrasound and Compact MRI for the Assessment of Cardiac Function in Mice
Authors: Coulthard Tonya , Wu Jun
Published: July 2021

Introduction:
Ultrasound (US) is a standard for the assessment of cardiovascular function. Pre-clinically, this is due the modality’s translational potential, its high temporal resolution and the absence of infrastructure. Recent advancements in novel high-performance compact MRI has made cardiac MRI a more accessible technique for assessing a variety of pathologies in murine models of CVD while reducing the complexity and costs traditionally associated with superconducting MRI systems. These developments may allow compact MRI to overcome some of the limitations of US meanwhile producing viable solutions for quantification along with new capabilities for cardiovascular biomarker assessment.

Hypothesis:
This study explores the relative capabilities of novel compact high-performance MRI compared to HFU for phenotypic analysis of mouse models of CVD.

Methods:
Four normal and one induced myocardial infarct C57BL/6 mice were imaged using both US and MRI. MI mice were prepared by ligation of the left anterior descending artery. In order to assess the ability of each modality to qualitatively and quantitatively characterize cardiac function, CINE loops of long axis and short axis slices were acquired with both systems and analyzed offline. In addition to traditional measures of cardiac function, strain analysis was performed using the HARP method for compact MRI and using speckle tracking for US. Finally, a cardiac MRI method for mapping and measuring infarct extent using gadolinium (Gd) contrast agents is described.

Results and Conclusions:
Although compact MRI has lower temporal resolution, when compared with US, it still provides many advantages in pre-clinical CVD imaging. In particular, because MRI signal is not attenuated by dense tissue or air, compact MRI can successfully generate artefact-free imaging of pathologies difficult or impossible to image with US, such as clearly visualizing and quantifying endo and epicardial borders. It also provides a whole body image and a “whole heart” image, making pre-clinical CV imaging easier for biologists and helping to reduce inter-operator variability. Finally, high sensitivity to Gd-based contrast agent with compact MRI enable new applications such as infarct quantification.
Abstract 106: Pluripotent Stem Cell Microrna-294 Induces Cardiomyocyte Proliferation and Augments Cardiac Function After Myocardial Infarction
Authors: Khan Mohsin , Nickoloff Emily , Johnson Jennifer , Verma Suresh , Ibetti Jessica , Garikipati Venkata N , Zhou Jibin , Benedict Cynthia , Koch Walter J , Kishore Raj
Published: July 2021

Rationale:
Embryonic heart is characteristic of rapidly dividing cardiomyocytes that give rise to sufficient numbers required to build a working myocardium. In contrast, cardiomyocytes retain some proliferative capacity in the neonates but lose most of it in adulthood. Embryonic stem cell cycle (ESCC) miRs are a class of microRNAs regulating the unique cell cycle of ESCs and their characteristic pluripotency. Nevertheless, expression of miR-294, a member of the ESCC miRs is lost during developmental transitions from the ESCs to mature cells. Effect of miR-294 to induce cardiac proliferation and heart function has not been previously studied.

Objective:
To determine whether miR-294 drives cardiomyocyte cell cycle reentry leading to augmentation of cardiac function after myocardial infarction.

Methods and Results:
miR expression analysis in the heart during development revealed elevated levels of miR-294 in the prenatal stages while the expression was lost in the neonates and adults as confirmed by qRT-PCR. Neonatal ventricular cardiomyocytes (NRVMs) were treated with miR-294 mimic to determine the effect on proliferation and cell cycle. Elevated mRNA levels of cyclins A2, E1, CDK2 together with c-myc, E2F1 and E2F3 was observed in NRVMs treated with 25nM mimic for miR-294. Additionally, miR-294 treated NRVMs showed in AKT phosphorylation along with enhanced protein levels of cyclin D1 and E2F1. Increased expression of p-histone 3, Ki67 and Aurora B kinase (G2/M) was confirmed by immunocytochemistry in NRVMs after miR-294 treatment compared to control cells. Administration of miR-294 in mice subjected to myocardial infarction demonstrated augmentation of cardiac function in mice receiving miR-294 8 weeks after injury. Increase myocyte proliferation was observed in the heart after miR-294 treatment as analyzed by BrdU uptake, p-Histone 3 and Aurora B expression by immunostaining. Concurrently, a decrease in infarct size along with decreased apoptosis was observed in the miR-294 hearts compared to the control.

Conclusion:
Ectopic expression of miR-294 recapitulates embryonic signaling and enhances cardiomyocyte ability to proliferate and reenter the cell cycle leading to augmented cardiac function in mice after myocardial infarction.
Abstract 109: Characterization of Secretome From C-kit+ Cells Derived From Neonate and Adult Heart Patients
Authors: Sharma Sudhish , Mishra Rachana , Goo Young A , Wehman Brody P , Guo Yin , Datla Srinivasa R , Balachandran Keerti , Siddiqi Osama T , Kaushal Sunjay
Published: July 2021

Background:
Despite the regenerative benefit reported in recent human clinical trials (SCIPIO trial) using human cardiac stem cells (CSCs) (identified by ckit+CD45-/lin-), low retention and differentiation of transplanted hCSCs into cardiomyocytes are insufficient to explain the myocardial regeneration. This discrepancy has led to the paracrine hypothesis of stem cell action that indicates communication between newly transplanted stem cells and reparative native cells, essentially jumpstarting the repair process.

Methods:
We derived CSCs from the biopsies of right atrial appendage (RAA) of neonatal human subjects (nCSCs, age < 2 months) and from adult human subjects (aCSCs, age >50 years). CSCs were grown upto 90% confluency and incubated with basal medium (Ham’s/F12) for 72 hours. Conditioned medium (CM) was collected and further analyzed.

Results:
The expression levels of various key cytokines were analyzed using ELISA and data showed nCSCs secrete higher levels of the angiogenic cytokines like SDF1, VEGF-A, ANG, SCF, PDGB, and FGF2 compared to over 20 other cytokines we examined as compared to aCSCs. CM derived from nCSCs showed more angiogeneic potential as compared to CM derived from aCSCs as analyzed by HuVEC tube assay formation. We also did a comparative analysis of CM from nCSCs and aCSCs using mass spectrometry. Molecular distribution of lead proteins revealed that the yhCSCs has more angiogenic, anti-inflammatory and proliferative proteins while ahCSCs have more of apoptotic proteins

Conclusion:
These data provide preliminary evidence that there is a difference in the cytokine secretion by the nCSCs as compared to aCSCs and implicates highly expressed cytokines as the key to myocardial recovery.
Abstract 111: NEDD8 Ultimate Buster-1 Long (NUB1L) Protein Regulates Atypical Neddylation and Protects Against Myocardial Ischemia-reperfusion Injury
Authors: Li Jie , Ma Wenxia , Li Huizhong , Hou Ning , Wang Xuejun , Kim Il-man , Li Faqian , Su Huabo
Published: July 2021

Neddylation is a ubiquitination-like pathway that covalently conjugates NEDD8 to target proteins and involves in diverse cellular processes. Under stress conditions, NEDD8 forms a chain or mixes with ubiquitin to modify protein substrates in NEDD8 conjugating enzymes-independent manner (atypical neddylation). The functional consequence of atypical neddylation remains unexplored in any cell types including cardiomyocytes. Here we report that increased neddylated proteins were observed in desmin-related cardiomyopathic (DRC) mouse hearts, mouse hearts subjected to myocardial ischemia-reperfusion (I/R) and human failing hearts. In cultured cardiomyocytes, multiple cellular stresses induced atypical neddylation, which was attenuated by NUB1L overexpression but exaggerated by loss of NUB1L, revealing NUB1L as a negative regulator of atypical neddylation. Activation of atypical neddylation by forced expression of NEDD8 accumulated a proteasome surrogate substrate GFPu, while suppression of atypical neddylation by NUB1L overexpression enhanced the degradation of GFPu and a DRC-linking misfolded protein. NUB1L is necessary and sufficient to protect cardiomyocytes against proteotoxic stress-induced cell injury. In vivo, cardiac-specific overexpression of NUB1L (NUB1L-O/E) in mice dose-dependently reduced neddylated proteins and facilitated the degradation of the proteasome surrogate substrate. NUB1L-O/E mice displayed no discernible cardiac structural and functional abnormality at baseline, but exihibted reduced apoptotic cardiomyocytes, limited infarct sizes and preserved cardiac function in response to I/R. We therefore conclude that NUB1L suppresses atypical neddylation, enhances proteasome proteolytic function and protects against myocardial I/R injury. Targeting atypical neddylation could be a novel therapeutic strategy to treat cardiac ischemic cardiomyopathy.
Abstract 114: AdipoRon, the First Orally Active Adiponectin Receptor Activator, Attenuates Post-ischemic Myocardial Apoptosis via AMPK Activation and Oxidative Stress Reduction
Authors: Wang Yajing , Lau Wayne , Du Yuanhui , Gao Erhe , Koch Walter , Ma Xinliang (Xin)
Published: July 2021

Adiponectin (APN) is a cardioprotective molecule. Its reduction in diabetes exacerbates myocardial ischemia/reperfusion (MI/R) injury. Although APN administration in animals attenuates MI/R injury, multiple factors limit its clinical application. The current study investigated whether AdipoRon, the first orally active molecule that binds APN receptors, may protect the heart against MI/R injury, and if so, to delineate the involved mechanisms. Wild type (WT) or gene manipulated mice were treated with vehicle or AdipoRon (50 mg/kg, 10 minutes prior to MI) and subjected to MI/R (30 minutes/3-24 hours). Oral administration of AdipoRon to WT mice significantly improved cardiac function (P<0.01) and reduced infarct size (P<0.01). At cellular level, AdipoRon attenuated post-ischemic cardiomyocyte apoptosis determined by DNA ladder formation, TUNEL staining, and caspase-3 activation (P<0.01). MI/R-induced apoptotic cell death was significantly enhanced in APN deficient mice (APNKO), and AdipoRon attenuated MI/R injury to the same degree observed in WT mice, indicating the pathological exageration caused by APN deficience can be rescured by a small molecule APN receptor activator. Cardiomyocyte-specific inhibition of AMPK (AMPK-DN), the most significant molecule mediating APN’s metabolic regulatory function, also increased post-ischemic apoptosis. Interestingly, AdipoRon’s anti-apoptotic action was partially inhibited, but not lost in AMPK-DN mice, indicating the anti-apoptotic effect of AdipoRon cannot be completely atributed to AMPK activation. AdipoRon significantly attenuated post-ischemic oxidative stress in an AMPK-independent fashion, as evidenced by reduced NADPH oxidase expression and superoxide production in both WT and AMPK-DN mice. Collectively, these results demonstrate for the first time that AdipoRon, an orally active APN receptor activator, effectively attenuated post-ischemic cardiac injury, supporting APN receptor agonists as a promising novel therapeutic approach treating cardiovascular complications caused by obesity-related disorders such as type 2 diabetes.
Abstract 12: Physiological Role of Endogenous Adult Cardiac Colony-forming Unit Fibroblasts
Authors: Forte Elvira , Janbandhu Vaibhao , Asli Naisana S , Cornwell James , Abeygunawardena Dhanushi , Xaymardan Munira , Harvey Richard P
Published: July 2021

Colony-forming unit fibroblasts (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), have been identified in different tissues. CFU-Fs are multipotent progenitor cells, with a supportive paracrine and immunomodulatory function. They share the expression of surface antigens, are capable of self-renewal, and to differentiate into osteogenic, adipogenic and chondrogenic lineages in vitro. Our laboratory has isolated, for the first time, CFU-Fs from the adult murine heart. These cells are included within the Sca1+ PDGFRα+ CD31- fraction of interstitial cells, mainly associated with microvessels and the perivascular adventitial niche of larger vessels. Here we show that resident cCFU-Fs, are quiescent in homeostatic conditions, relatively more hypoxic and have a lower metabolic profile compared to other interstitial cells. Treatment with PDGF-AB ligand stimulates cCFU-Fs to exit the quiescent state, potentially making them more responsive to mitogenic and differentiative factors. In a mouse with a H2B-eGFP fusion gene knocked-in the Pdgfrα locus, cCFU-Fs lay in the GFPhigh population. After myocardial infarction, a GFPmed population appears, which has partially lost the expression of Sca1 and PDGFRα proteins, and consists largely of activated myofibroblasts. Likely derived from cCFU-Fs or related stromal fraction, GFPmed cells are significantly reduced by inhibiting PDGFRα signalling and increased by systemic PDGF-AB treatment. Despite augmenting the GFPmed myofibroblasts-like population, short-term PDGF-AB treatment is beneficial, leading to reduced scar, and increased vascular density and ejection fraction. A crucial factor in cardiac repair is the fine balance between pro-inflammatory (type1) and pro-reparative/pro-angiogenic (type2) immune responses, which can be regulated by MSCs. interestingly PDGF-AB treatment seems to be associated with an earlier resolution of the type1 response at 5 days after MI, compared to controls. We hypothesize that the beneficial effects of short term PDGF-AB treatment are due to early activation of cCFU-Fs and stromal cells from quiescence, leading to enhanced pro-angiogenic, anti-apoptotic and immunomodulatory impacts.
Abstract 131: A microRNA Targets the Epigenetic Reader BRD4 to Suppress Transcription Elongation in the Heart
Authors: Stratton Matthew S , Ferguson Bradley S , Lin Charles Y , Anand Priti , Wickers Sean T , Tatman Philip D , Bradner James E , Haldar Saptarsi M , McKinsey Timothy A
Published: July 2021

BRD4 is a member of the BET family of proteins, which contain tandem reader domains (bromodomains) that bind to acetylated histone tails within chromatin. We recently demonstrated that JQ1, a small molecule that prevents BRD4-chromatin interaction, potently blocks pathological cardiac hypertrophy and improves cardiac function in pre-clinical models. BRD4 functions as a nodal regulator of the transcriptional program for cardiac hypertrophy by recruiting P-TEFb to gene regulatory elements, resulting in phosphorylation of RNA polymerase II (Pol II), and subsequent transcription elongation. Here, we describe a signal-dependent mechanism for regulation of BRD4 in the heart. BRD4 protein expression is dramatically upregulated in cardiomyocytes exposed to cues for pathological, but not physiological, hypertrophy. In unstimulated cardiomyocytes, BRD4 protein expression is maintained at low levels by a microRNA, which directly targets the 3’ UTR of the BRD4 transcript, thereby preventing translation of this acetyl-lysine reader. In response to signals for hypertrophy, expression of the microRNA is down regulated through a histone deacetylase (HDAC)-dependent mechanism, resulting in robust induction of BRD4 protein synthesis. Whole genome analyses establish a crucial role for the microRNA in the control of Pol II elongation in the heart via BRD4 down regulation. These findings define a novel chromatin signaling axis that can be targeted at multiple levels (epigenetic reader, microRNA or HDAC) for the treatment of heart failure.
Abstract 14: Impact of Gata4, Mef2c and Tbx5 Stoichiometry on Epigenetic Status of Direct Cardiac Reprogramming
Authors: Qian Li , Liu Ziqing , Zheng Michael , Liu Jiandong
Published: July 2021

Reprogramming from fibroblasts into induced cardiomyocytes (iCMs) offers alternative strategies for cardiac disease modeling and cardiac regeneration. Cellular reprogramming is closely associated with global re-patterning of the epigenetic landscape. The reprogramming cells must overcome the epigenetic barriers to acquire the target cell-like epigenetic pattern. We have recently demonstrated that stoichiometry of iCM reprogramming factors Gata4 (G), Mef2c (M) and Tbx5 (T) has profound impact on reprogramming efficiency and quality, yet the underlying mechanism remains unclear. Here, we demonstrate that the optimal stoichiometry combination MGT resulted in a reduced binding of the repressive marker histone 3 lysine 27 trimethlytion (H3K27me3) and an enhanced binding of the active marker histone 3 lysine 4 trimethlytion (H3K4me3) on the cardiac gene loci, compared to the least optimal combination GTM. We found that this epigenetic change occurred as early as day 3 when cardiac marker genes started to be induced in the reprogramming fibroblasts. Additionally, we determined the DNA methylation status in two cardiac gene loci during MGT and GTM induced reprogramming. Subsets of the CpG islands in these promoters were significantly more demethylated in MGT than GMT-transduced cells. We thus propose these CpGs islands as the “regulatory” CpGs critical for iCM reprogramming. In conclusion, we demonstrated that stoichiometry of G, M, T influences the epigenetic status of the iCM cells, suggesting a potential mechanism of how stoichiometry of G,M,T influences reprogramming.
Abstract 154: Allogeneic Cardiosphere-derived Cells From an Aged Donor Elicit Long-term Improvements in Left Ventricular Function and Myocyte Proliferation in a Mini-swine Model of Chronic Myocardial Ischemia
Authors: Weil Brian R , Suzuki Gen , Leiker Merced M , Goelz Andrew , Canty John M
Published: July 2021

Objective:
Virtually all large animal studies of cell-based therapy have employed young donor-recipient pairs. Since aging may impact the reparative ability of allogeneic cardiosphere-derived cells (CDCs), we tested whether CDCs from an aged donor promote functional repair of ischemic myocardium using a mini-swine model that enabled serial assessment of left ventricular (LV) function over an extended follow-up period.

Methods:
Immunosuppressed mini-swine (cyclosporine 100 mg/day) with a chronic (4-months) LAD stenosis were untreated (n=8) or received 20 million allogeneic aged CDCs (n=10). Cells were cultivated from a 9-year old mini-pig and infused into the 3 major coronary arteries under continuous flow (1 million cells/min). LV function was assessed by echocardiography at baseline and over a 3-month follow-up period, at which time histological assessment of myocyte morphometry, proliferation (Ki67), and cell retention (Y-FISH) was performed.

Results:
Wall thickening (WT) of the ischemic LAD region was impaired at baseline (LAD: 42.7 ± 2.1% vs. Remote: 81.4 ± 4.8%, p<0.01) and not different between treatment groups. One month later, LAD WT improved in aged CDC-treated animals (from 41.0 ± 3.1 to 53.0 ± 1.7%, p=0.01) but remained depressed in untreated animals (from 45.1 ± 2.8% to 44.9 ± 2.6%, p=0.97). Extended follow-up revealed that LAD WT continued to improve, reaching 60.2 ± 1.6% (p=0.01) 3-months after CDC treatment. Histological analyses demonstrated morphometric changes consistent with myocyte regeneration, including an increase in myocyte nuclear density (1231 ± 34 vs. 1094 ± 34 nuclei/mm
2
, p=0.02) and reduction in myocyte diameter (13.9 ± 0.2 vs. 14.5 ± 0.3 μm, p=0.05). Increases in Ki67
+
myocytes (468 ± 92 vs. 199 ± 32 nuclei/10
6
myocyte nuclei, p=0.02) and rare Y
+
cells in sex-mismatched recipients (0.5 ± 0.2 cells/cm
2
) supported endogenous myocyte proliferation as the primary source of new myocytes.

Conclusion:
CDCs from an aged donor retain their reparative capacity to promote functional improvement and increase myocyte number following global intracoronary infusion. The progressive improvement in LV function 3-months after treatment with ongoing myocyte proliferation supports a long-lasting benefit after a single infusion.
Abstract 155: C-kit+ Cardiac Progenitor Cells Contain a Subpopulation of Slowly Adherent Cells With Higher Cardiomyogenic and Pro-angiogenic Profile
Authors: Ghafghazi Shahab , Ponnapureddy Rakesh , Solanki Mitesh , Sharma Sorabh , Kayathi Pramod , Sharma Bhavna , Bolli Roberto , Wysoczynski Marcin
Published: July 2021

Background:
Isolation of a pure population of cardiomyogenic cardiac stem cells (CPCs) has been the Holy-Grail in cardiac regeneration. C-kit, a cell surface receptor, has been proposed as a marker of CPCs and the result of multiple preclinical studies and a phase I clinical trial has been encouraging. Nonetheless, optimization of isolation methods is desirable given the heterogeneity of c-kit positive CPCs. We hypothesize that isolation of c-kit positive CPCs based on differential adhesion identifies a superior population of cells.

Methods:
Non-adherent cells from digested murine hearts were transferred to successive flasks at 2, 4, 24, 48 and 72 hours. Hence, 5 fractions of cells were isolated based on their adhesion profile and labeled rapidly-adherent (1 and 2) and slowly-adherent (3 to 5). Cells were then expanded and sorted based on c-kit expression and expanded to seven passages. Cells were analyzed for surface markers and cardiomyogenic and pluripotent expression profile by flow-cytometry and real-time polymerase chain reaction. Differentiation potential of the two fractions was examined and their paracrine profile compared using human umbilical vein endothelial cells and neonatal rat cardiomyocytes.

Results:
C-kit expression was maintained in the slowly-adherent fraction while precipitously dropping in the rapidly-adherent one. In addition, the former expressed lower levels of mesenchymal/fibroblast (CD90.2) and macrophage (CD11b/CD45) markers and higher levels of pluripotent (Oct-4, Nanog, Dppa-3, Rif) and cardiac markers (Nkx2.5, Gata4, Mybpc3 and cTnI) comparing to the latter. Finally, slowly-adherent CPCs had superior paracrine profile compared to rapidly-adherent one.

Conclusion:
C-kit positive CPCs cells isolated from murine myocardium are heterogeneous and better isolation methods are required to enrich a purer population of cardiomyogenic stem cells. In that vein, isolation of c-kit positive CPCs based on their adhesion profile identifies a subpopulation of cells capable of maintaining c-kit positivity and superior in terms of cariomyogenic and pro-angiogenic potential.
Abstract 157: Unique Features of Cortical Bone Stem Cells Associated With Enhanced Cardiac Repair
Authors: Mohsin Sadia , Troupes Constantine D , Sharp Thomas E , Starosta Timothy , Agra Elorm J , Smith Shavon , Kubo Hajime , Berretta Remus M , Houser Steven R
Published: July 2021

Rationale:
Adoptive transfer of bone marrow and cardiac derived stem cells (CDCs) into failing human hearts has been shown to be safe, yet these cells have only induced modest improvements after myocardial infarction (MI). Recently we have shown in a mouse model that cortical bone derived stem cells (CBSCs) induced a greater enhancement of cardiac function after MI through enhanced paracrine signaling and transdifferentiation of CBSCs into new cardiac tissue. However, the reparative potential of CBSCs relative to other stem cell types including bone marrow derived mesenchymal stem cells (MSCs) and CDCs is not known.

Objective:
To characterize surface marker expression, proliferation, survival, migration and differentiation capacity of swine CBSCs relative to MSCs and CDCs.

Methods and Results:
CBSCs, MSCs and CDCs were isolated from Gottingen miniswine. CBSCs were morphologically distinct from MSCs and CDCs, with differences in length to width ratio and overall cell surface area. Cell surface marker profiling using RT-PCR analysis revealed that CBSCs express some of the classical MSC markers such as CD106, CD271, CD105, CD90 and CD29 and are negative for CD45 and CD11-b. CBSCs had an enhanced proliferation capacity versus CDCs and MSCs, measured by CyQuant assay. Concurrently CBSCs had significantly decreased population-doubling time (3.57 and 1.26 fold decrease) as compared to MSC and CDCs. CBSCs exhibit enhanced survival after exposure to apoptotic stimuli as compared to MSCs measured by Annexin-V staining. A significantly greater % of CBSCs expressed markers of cardiac lineage commitment when exposed to dexamethasone than did CDCs or MSCs. Markers of cardiac lineages including GATA-4, α SMA, Troponin T, sm22 were measured with RT-PCR and immunocytochemistry.

Conclusion:
CBSCs have enhanced proliferative, survival capacity and cardiac lineage commitment versus CDCs and MSCs that could account for their enhanced effects on cardiac regeneration after myocardial infarction.