bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 411: Discovery and Cardioprotective Effects of the First Non-peptide Agonists of Prokineticin Receptor-1
Authors: Nebigil Canan G , Gasser Adeline , Brogi Simone , Tafi Andrea , Kurose Hitoshi , Désaubry Laurent
Published: July 2021

Prokineticins are angiogenic hormones that activate two G protein-coupled receptors: PKR1 and PKR2. PKR1 has emerged as a critical mediator of cardiovascular homeostasis and cardioprotection. Identification of non-peptide PKR1 agonists that contribute to myocardial repair and collateral vessel growth hold promises for treatment of heart diseases. Through a combination of in silico studies, medicinal chemistry, and pharmacological profiling approaches, we designed, synthesized, and characterized the first PKR1 agonists, demonstrating their cardioprotective activity against myocardial infarction (MI) in mice. Based on high throughput docking protocol, 250,000 compounds were computationally screened for putative PKR1 agonistic activity, using a homology model, and 10 virtual hits were pharmacologically evaluated. One hit internalizes PKR1, increases calcium release and activates ERK and Akt kinases. Among the 30 derivatives of the hit compound, the most potent derivative, IS20, was confirmed for its selectivity and specificity through genetic gain- and loss-of-function of PKR1. Importantly, IS20 prevented cardiac lesion formation and improved cardiac function after MI in mice, promoting proliferation of cardiac progenitor cells and neovasculogenesis. The preclinical investigation of the first PKR1 agonists provides a novel approach to promote cardiac neovasculogenesis after MI.
Abstract 422: Activation of the p38 Branch of Mitogen Activated Protein Kinase Pathway Stimulates Proteasome Proteolytic Function
Authors: Wang Xuejun , Wang Changhua , Terpstra Erin J , Wang Yibin , Wang Xuejun “XJ”
Published: July 2021

Ubiquitin-proteasome system (UPS) dysfunction is associated with development of most heart diseases. Proteasomal functional insufficiency has been experimentally demonstrated to play an important role in cardiac pathogenesis. Hence, enhancing proteasome function is potentially a powerful new strategy for therapeutic exploration but this research area is hindered critically by poor understanding of the regulation of cellular proteasomes. Here, we sought to determine whether and how the p38 branch of mitogen activated protein kinase (MAPK) regulates the UPS. GFPu is a rationally modified green fluorescence protein (GFP) proven to be a UPS-specific substrate. In both an HEK293-GFPu/RFP stable cell line and cultured neonatal rat ventricular myocytes (NRVMs), serum withdrawal activated p38 and decreased GFPu/RFP protein ratios. The decrease was abolished by p38 inhibitor (SB230580). Similar effects were also observed when p38 was activated by anisomycin. Cycloheximide (CHX) chase assays showed that p38 activation by either anisomycin treatment or MKK3 overexpression significantly shortened the half-life of GFPu proteins. These compelling data demonstrate for the first time that p38 stimulates UPS performance. To pinpoint the step of the UPS pathway which p38 acts on, we examined the level of total ubiquitin conjugates and proteasomal peptidase activity. Activation of p38 via various means failed to increase the level of total ubiquitin conjugates but was able to increase significantly proteasomal chymotrypsin-like activity in cultured NRVMs and this increase was remarkably blunted by SB230580. The altered proteasome activity was not associated with discernible changes in the protein abundance of representative subunits of the 19S (Rpt6, Rpn2) and 20S proteasomes (α5, β5, β7); however, 2-dimensional PAGE followed by immunoblot showed that the isoelectric point (pI) of a fraction of β5, but not α2, subunits of the 20S proteasome was shifted to the acidic side, consistent with increased phosphorylation, by MKK3 overexpression in cultured NRVMs. It is thus demonstrated for the first time that p38 MAPK stimulates proteasome proteolytic function and it achieves such effect likely through post-translational modifications of the proteasome.
Abstract 428: DNA Methylation of ASC is Associated with Decreased ASC and IL-1β Expression in Heart Failure
Authors: Butts Brittany , Butler Javed
Published: July 2021

Introduction:
Heart failure (HF) is associated with formation and activation of inflammasome, a complex of intracellular interaction proteins that trigger maturation of inflammatory cytokines to initiate inflammatory response. ASC, a vital component of the inflammasome, is controlled through epigenetic modification via methylation of CpG islands surrounding exon 1.

Methods:
To assess the relationships between DNA methylation of ASC, ASC expression, and inflammatory cytokines IL-1β and IL-18 in HF, stored samples from 155 chronic HF patients (age 56.9±12.0 yr, 64% male, 47% black, and ejection fraction 29.9±14.9) were analyzed. DNA extracted from PMBCs were analyzed by pyrosequencing for percent methylation of seven CpG sites in the intron region preceding exon 1 of the ASC gene. ASC mRNA was quantified via real-time PCR and analyzed as the ratio ASC:GAPDH. Serum ASC, IL-1β, and IL-18 were measured by ELISA.

Results:
Higher ASC methylation was associated with lower ASC mRNA (r=0-.328, p<0.001) and protein (r=-.464, p<0.001) expression. Lower ASC mRNA expression was associated with lower ASC protein expression (r=0.494, p<0.001). Decreased IL-1β expression was associated with higher ASC methylation (r=-.424, p=0.005) and lower ASC mRNA (r=.619, p<0.001) and ASC protein (r=.433, p<0.001). IL-18 expression was not significantly associated with ASC methylation or expression.

Conclusions:
Increased ASC methylation was associated with lower IL-1β, likely via decreased ASC gene expression. As ASC is required for inflammasome activation of IL-1β, this study implicates the inflammasome pathway as a driver of inflammation in HF, proving a potential target for novel interventions.
Abstract 429: MicroRNA Control of the Pacemaker Channel and Training-induced Bradycardia
Authors: D’Souza Alicia , Gill Eleanor , Cox Charlotte , Dobrzynski Halina , Cartwright Elizabeth , Oceandy Delvac , Boyett Mark R
Published: July 2021

Background:
Athletes are prone to bradyarrythmias and have a higher-than-normal incidence of pacemaker implantation but the underlying mechanisms are poorly understood. In previous work we demonstrated a training-induced electrical remodelling of the sinoatrial node (SAN), including a down regulation of the key pacemaker channel HCN4 that carries the pacemaker current
I
f
. Here we investigated post-transcriptional regulation of HCN4 by microRNAs (miRNAs) in the trained SN.

Methods and Results:
Mice subjected to 60-min swimming twice daily for 28 days (TM) were compared to parallel sedentary mice (SM).TM were bradycardic (cycle length
in vivo
: SM, 81±1.3 ms; TM, 102±3 ms; n=6,
p
<0.05). The cycle length of the isolated, denervated SAN (intrinsic heart rate) was also prolonged by exercise training (SM, 110±3 ms; TM, 150±5 ms; n=7,
p
<0.05). Deep sequencing for miRNAs on SAN punch biopsies revealed 10 differentially expressed miRNAs in TM
vs
SM (FDR adjusted
P
<0.05); 8 miRNAs were upregulated, 2 were downregulated. These findings were further confirmed by real-time PCR. We used the computation prediction tools
Targetscan
and
Ingenuity Pathway Analysis
to identify miR-423-5p and miR-486-3p as putative miRNAs participating in the post-transcriptional repression of HCN4 in the trained SAN. Both miRNAs had conserved seed sequences in the HCN4 3’UTR. To validate whether these miRNAs directly recognise the 3’UTR of HCN4, they were co-transfected with a construct containing HCN4 3’UTR fused downstream to a luciferase coding sequence in H9c2 cells. Transient transfection of precursor miR-423-5p and miR-486-3p decreased HCN4 3’UTR luciferase reporter activity in H9C2 cells by 47% and 22% respectively, compared with unaltered luciferase activity on co-transfection with control miR or miR-27a.

Conclusions:
This is the first report of miRNA dysregulation in the trained SAN. We describe a novel molecular mechanism whereby an altered miRNA profile in the trained SAN promotes HCN4 downregulation and altered sinus automaticity. Data suggest miR-423-5p as a potential therapeutic target for bradyarrythmias in the athlete.
Abstract 45: Systemic Illness in Ece1 Ablated Adult Mice
Authors: Kristianto Jasmin , Johnson Michael , Radcliff Abigail , Koch Jill , Hacker Timothy , Patel Forum , Zastrow Ryley , Wang Xiaohu , Yuan Baozhi , Blank Robert
Published: July 2021

Endothelin converting enzyme-1 (ECE1) catalyzes the conversion of inactive big endothelin 1 (ET1) to active ET1. Homozygous
Ece1
knock out (KO) mice die
in utero
or at birth, displaying multiple abnormalities including mandibular hypoplasia and cardiac outflow tract malformations, in spite of the presence of ample tissue ET1. However, increased ECE1 activity and circulating and/or tissue ET1 are associated with many adult cardiovascular diseases, including idiopathic pulmonary fibrosis (IPF), a chronic and fatal lung disease. There is an apparent paradox between the need for ET1 in development and its harmful effects in adult disease. Therefore, our lab developed a conditional
Ece1
KO mouse, in which
Ece1
is ablated following tamoxifen (tam) treatment. We hypothesized that ECE1 serves to localize ET1 signals to specific cell populations and is essential in normal adult physiology. We studied the following groups: mice given vehicle rather than tam, mice lacking tam-inducible Cre recombinase, mice harboring a normal
Ece1
allele (

Ece1
+/flox

), and the experimental animals (Cre

Ece1
-/flox

). Mice were treated with vehicle or tam at 8-9 weeks of age. Cre

Ece1
-/flox

mice showed 85-100% mRNA knock-down efficiency 8 weeks after tam treatment. By 17 weeks of age,

Cre

Ece1
-/flox

mice have tachypnea, decreased activity, and weight loss, requiring euthanasia for humane considerations. They display depleted adipose tissue mass compared to controls. By two weeks after treatment, Cre

Ece1
-/flox

mice had lower blood pressure relative to controls, which persisted until euthanasia at 17-20 weeks old (p=0.004). Between 17-20 weeks of age, most of Cre

Ece1
-/flox

mice develop pectus excavatum, enlarged right hearts and have reduced stroke volume and cardiac output as analyzed by echocardiography. Histological examination revealed eosinophilic crystalline pneumonia and increased collagen in the lung and heart. These findings are consistent with development of IPF in the experimental mice. Our findings show that
Ece1
ablation in post-natal animal results in a severe cardiorespiratory disease, suggesting that ectopic activation of ET1 by other tissue proteases is the primary mechanism underlying the association of increased ET1 signaling in disease states.
Abstract 48: Clonal Analysis of Cardiac Cell Generation During Cardiac Development and Injury
Authors: Sereti Konstantina Ioanna , Kamran Paniz , Zhao Peng , Ranjbarvaziri Sara , Engel James , Mikkola Hanna , Ardehali Reza
Published: July 2021

Accumulating evidence supports limited regenerative potential of the mammalian heart. However, this endogenous regenerative capacity significantly decreases with age and it is not sufficient to replenish the lost myocardium following injury in the adult. Both resident cardiac stem/progenitor cells and mature cardiomyocytes have been proposed to contribute to cardiac tissue generation. Understanding the molecular and cellular mechanisms governing cardiac cell formation is imperative towards the development of novel therapeutic strategies for cardiac regeneration. Hypothesis: Cardiac growth occurs primarily through progenitor cells and to a lesser extent through cardiomyocyte proliferation. Nkx2.5+ cells are the predominant population driving cardiac growth during development and may represent candidate progenitors for cardiac regeneration. Moreover, a subset of “proliferating” αMHC+ cardiomyocytes may also contribute to cardiac growth during early embryonic development and the first week of life. Materials and methods: We performed clonal analysis using a multicolor reporter system (Rainbow) that allows labeling of single cells with one of three fluorescent proteins and retrospective analysis of their progeny. Rainbow mice were crossed to βactinCreER, αMHCCreER, Nkx2.5CreER and Rosa26CreER mice. Tamoxifen was administered at E9.5 or E12.5 and analysis was performed at P1, P7, P15 and P30. Results: We observed significant clonal expansion in βactinCreER;Rainbow and Nkx2.5CreER;Rainbow hearts while αMHCCreER;Rainbow hearts exhibited clones comprising of smaller cell number. We also found that αMHC positive cells lose their proliferative capacity from E9.5 to E12.5, whereas cells expressing Nkx2.5 continue to clonally expand during that same time period. Finally, we demonstrated that αMHC+ cardiomyocyte proliferation is reactivated following myocardial injury soon after birth. Conclusion: Our data support that clonal dominance of progenitor cells promotes cardiac development, while cardiomyocyte proliferation contributes to a lesser extent early in development and postnatally.
Abstract 5: Novel Tools for Identification and Purification of Cardiomyocytes, Fibroblasts and Endothelial Cells From Neonatal Mouse and Rat Hearts
Authors: Eckardt Dominik , Kernbach Manuel , Czichowski Vera , Hintzen Christoph , Bosio Andreas
Published: July 2021

Although isolation of vital cardiac cells from neonatal hearts is one of the most used experimental model in cardiac research, the manual dissociation of neonatal hearts is a laborious and difficult-to-standardize procedure. To shorten sample processing time and increase reproducibility, we developed a fully automated dissociation process followed by magnetic purification of cardiomyocytes (CMs), cardiac fibroblasts (CFs) and endothelial cells (ECs). Neonatal hearts were dissociated within 1 h resulting in high yields of single cells with excellent viability. Depending on age and species following cell ratios were recovered after dissociation: 50-70% CMs, 30-40% CFs, 10-20% ECs.

In order to purify CMs from dissociated neonatal hearts, we defined antibody cocktails enabling non-myocyte removal. Our depletion strategy allowed for the enrichment of CMs from whole hearts or preparations of heart chambers with purities of up to 98%. To enable simultaneous detection of CMs and subtypes, we developed recombinant antibodies against general CM markers like alpha Actinin, Myosin Heavy Chain or cardiac Troponin T as well as CM subtype-specific antibodies against MLC2a and MLC2v distinguishing between atrial and ventricular CMs respectively.

Besides, we developed magnetic enrichment strategies for the purification of CFs and ECs. Current purification strategies that are solely based on antibodies against EC surface markers do not result in sufficiently pure cardiac ECs. Therefore, we established a 2-step enrichment protocol allowing for the complete removal of contaminating fibroblasts and leukocytes. Our data indicate that this protocol significantly improves the purity of primary cardiac ECs. Functionality of ECs before and after enrichment was proven by Dil-Ac-LDL endocytosis.

Similarly, our magnetic enrichment protocol for cardiac fibroblasts revealed homogenous expression of fibroblast markers like Vimentin and Prolyl-Hydroxylase, but virtually no contaminations with CMs or ECs.

In summary, we established an automated protocol for dissociation of neonatal hearts enabling subsequent purification, characterization and cultivation of CMs, CFs and ECs which can readily be used for cell culture assays or to generate heart muscle models.
Abstract 57: First Induced Heart Failure Model in Adult Zebrafish by Chronic Isoproterenol Treatment
Authors: Kossack Mandy , Katus Hugo A , Most Patrick , Hassel David
Published: July 2021

Impaired β-adrenergic signal transduction is a common molecular alteration found in heart failure (HF). Chronic catecholamine release and overstimulation of β-adrenergic receptors (β-ARs) in the failing heart results in their desensitization, largely mediated through upregulation of the G-protein coupled receptor kinase 2 (GRK2), and consequently to the progression into HF. Chronic activation of β-ARs by isoproterenol (iso) infusion efficiently induces HF in mice, while therapeutic targeting of GRK2 in HF animal models preserves cardiac function, highlighting their significance in HF progression.

Zebrafish represents an established model to evaluate genetic causes of HF and to screen for novel therapeutic targets. However, the contribution of the β-A system in zebrafish models of HF is not known. We here systematically analyzed the effect of iso on heart function in larval and adult zebrafish.

Larvae first responded to iso with 3 days of age (d). Here, β-AR stimulation resulted in the activation of conserved signaling components and in the induction of common stress responsive genes. Chronic β-AR stimulation for 5 days induced signs of HF accompanied by similar expression changes seen in mammals. As heart phenotypes are usually not analyzed in larval zebrafish beyond 3d, our data implicates that previous studies neglected a possible impact of β-AR signaling, particularly important when screening for therapeutic components.

Adult zebrafish recently emerged as an attractive cardiac model, especially for regenerative medicine. Echocardiography revealed that in adult zebrafish hearts, iso robustly enhanced cardiac function. Chronic β-AR stimulation for 14 days efficiently induced HF symptoms. Consistent with mammals, we found reduced expression of β-ARs and elevated expression of GRK2 and ANP. Additionally, these fish develop essential characteristics accompanied with HF, including increased cell death and elevated inflammation.

In conclusion, we show that β-AR function in zebrafish is comparable to that in mammals. Further, we present the first iso-induced HF model in adult zebrafish, thereby introducing adult zebrafish as a particularly valuable model to study the pathogenesis of HF and to test for novel therapeutic strategies to treat HF.
Abstract 72: Hydrogen Sulfide Prevents Myocardial Hypertrophy in a Klf5-dependent Manner
Authors: Meng Guoliang , Xie Liping , Ji Yong
Published: July 2021

Rationale:
H
2
S is a gasotransmitter that regulates multiple cardiovascular functions. Krüppel-like transcription factor (KLF) exerts diverse functions in the cardiovascular system.

Objectives:
The aim of present study was to investigate the effect of hydrogen sulfide (H
2
S) on myocardial hypertrophy.

Methods and results:
Myocardial samples of 22 patients with left ventricle hypertrophy were collected and underwent histological and molecular biological analysis. Spontaneously hypertensive rats (SHR) and neonatal rat cardiomyocytes were studied for functional and signaling response to GYY4137, a H
2
S-releasing compound. Expression of cystathionine -lyase (CSE), a main enzyme for H
2
S generation in human heart, decreased in human hypertrophic myocardium, while KLF5 expression increased. In SHR treated with GYY4137 for 4 weeks, myocardial hypertrophy was inhibited as evidenced by improvement in cardiac structural parameters, heart mass index, size of cardiac myocytes and expression of atrial natriuretic peptide (ANP). Levels of oxidative stress and phosphorylation of mitogen-activated protein kinases were also decreased after H
2
S treatment. H
2
S diminished expression of the KLF5 in myocardium of SHR and in neonatal rat cardiomyocytes rendered hypertrophy by angiotensin II (Ang II). H
2
S also inhibited ANP promoter activity and ANP expression in Ang II-induced neonatal rat cardiomyocyte hypertrophy, and these effects were suppressed by KLF5 knockdown. KLF5 promoter activity was increased by Ang II stimulation, and this was reversed by H
2
S. H
2
S also decreased activity of specificity protein-1 (SP-1) binding to the KLF5 promoter and attenuated KLF5 nuclear translocation by Ang II stimulation.

Conclusion:
H
2
S attenuated myocardial hypertrophy, which might be related to inhibiting oxidative stress and decreasing ANP transcription activity in a KLF5-dependent manner.
Abstract 8: Reprogramming of Adult Mammalian Cardiomyocytes Towards Proliferation and Regeneration Through Comparative Analyses of Epicardial and Myocardial Cells
Authors: Huang Guo , Judd Justin , Lovas Jonathan
Published: July 2021

Heart development and regeneration require an elegant balance of cell proliferation and differentiation. In both adult zebrafish and neonatal mouse cardiac regeneration, new cardiomyocytes are shown to originate from pre-existing cardiomyocytes through de-differentiation of mature cells to the immature progenitor cell state, with developmental gene program reactivation and cell cycle reentry. Interestingly, after adult mammalian cardiac injury, both cardiac muscle cells and the epicardial cells that envelope the heart reinitiate developmental gene programs. However, adult epicardial cells are able to proliferate following injury but adult cardiomyocytes are not capable. We investigated the genetic circuitry of mouse epicardial cells and myocardial cells in different developmental stages and in response to injury. Such comparative analyses revealed a group of ~50 candidate genes that may be responsible for the permanent cell cycle arrest of cardiomyocytes. We generated adenoviruses that express these candidate genes individually, and demonstrated that the mixed viral pool possessed a robust activity to promote proliferation of adult mouse cardiomyocytes. Our comparative approach and functional screens may lead to identification of the dormant genetic circuitry in adult mammalian heart that can be reactivated to drive robust cardiomyocyte proliferation and regeneration.
Abstract 90: Akt Mediated Mitochondrial Dysfunction Involves Mitochondrial Endothelial Nitric Oxide Synthase Translocation
Authors: Rafikov Ruslan , Rafikova Olga , Sun Xutong , Aggarwal Saurabh , Black Stephen M
Published: July 2021

Pulmonary arterial hypertension (PH) is a fatal disease characterized by uncontrolled pulmonary vascular cell proliferation. Mitochondrial dysfunction (MD) of pulmonary endothelial cells (EC) was shown to be one of the primary events implicated into proliferative, apoptosis resistant cell phenotype. However, the particular molecular mechanisms responsible for MD remain unclear. The development of PH in patients is associated with severe nitrosative stress, leading to post-translational protein modifications. Thus, we have recently found that Akt is susceptible to nitration of tyrosine Y350 residue. We hypothesize that nitration of Akt induces activation of Akt signaling and contributes to the development of MD. Nitrosative stress in EC was initiated by eNOS uncoupler ADMA or peroxynitrite donor SIN-1 and resulted in significant Akt nitration and activation (1.5±0.1 fold control; p=0.007; N=3-4), as well as activation of anti-apoptotic (BAD phosphorylation), and proliferative (mTOR phosphorylation) signaling cascades. Increased Akt signaling induced phosphorylation of eNOS at serine S615 (1.47±0.08 fold control, p=0.005, N=3) and S1177 (0.22±0.04 vs. 0.47±0.07, p=0.029, N=3). Phosphorylation of eNOS resulted in its translocation to mitochondria (3± 0.3 fold control; p=0.003; N=3) which, in turn, significantly decreased basal mitochondrial respiration (oxygen consumption rate, pmol/min: untransfected cells 1022±96 vs. mitochondrial targeted phospho-mimetic eNOS mutants S615D 102±9 and S1177D 117±7, p<0.001, N=4-6), perhaps due to previously reported inhibitory effect of NO on mitochondrial respiratory chain. Finally, we have created an anti-oxidant conjugated “shielding” peptide that, by shielding the Akt nitration site, is capable to prevent Akt activation. Indeed, pre-treatment with shielding peptide (100μg/ml, 30min) completely abolished SIN-1 induced nitration of Akt in EC. We conclude that Akt nitration may contribute to proliferative/apoptosis resistant EC phenotype through pathological activation of Akt signaling and Akt mediated mitochondrial eNOS translocation. Besides, our novel shielding peptide based therapeutic strategy opens new avenues in MD prevention.
Abstract 98: Embryonic Extracellular Matrix and Cell Fate Determination
Authors: Kren Stefan M , Chapman Christopher S , Loza Andrew , Garry Daniel J , Garry Mary G
Published: July 2021

Background
- The determination of cell fate during development is governed by intrinsic factors, but also by interaction with the milieu in which they reside. The extracellular matrix (ECM) from rapidly developing tissue should form a rich signaling environment for cellular proliferation and differentiation.

Hypothesis
- Murine embryonic ECM can be prepared by detergent decellularization that is morphologically preserved, biocompatible for cell culture, and at E13.5 substantial enough to permit vascular catheterization and recellularization by perfusion.

Methods and Results
- To test the contribution of embryonic extracellular matrix (ECM) to the determination of cell fate, we undertook isolation of ECM from developing murine embryos. Triton X-100 and SDS detergent decellularization were used to isolate ECM from E10.5 and E13.5 embryos. Acellularity was confirmed by pico-green DNA assay (98.7 % ± 0.95 of DNA removed compared to control), as well as the lack of visible nuclei by H & E and DAPI histology. The matrix scaffolds were washed thoroughly with PBS and culture media to return them to a biocompatible state. Murine embryonic stem cells (mESC) modified to express EGFP were cultured on the exterior or the interior of the ECM scaffolds. mESCs seeded on the exterior of the E10.5 scaffolds or perfused through the E13.5 umbilical vasculature were highly adherent and proliferative during the 17 day culture period as evidenced by fluorescent microscopy. Perfused mESCs exhibited engrafted in the heart, liver, and vascular conduit E13.5 matrix 2 days post-infusion. Histology confirmed the attachment and morphologic alteration of the cultured cells on the exterior of the E10.5 ECM and presence of the perfused cells in the E13.5 embryo matrix interior.

Conclusion
- Biocompatible, acellular morphologically preserved embryonic ECM can be extracted from E10.5 and E13.5 murine embryos. By E13.5 the structural integrity of the acellular matrix can sustain vascular perfusion for delivery of mESCs to internal organoid structures. These ECM preparations support the proliferation and maintenance of mESCs externally and internally.
Abstract 100: A MicroRNA Signature for Left Ventricular Reverse Remodeling in Chronic Systolic Heart Failure
Authors: Ziegler Olivia , Shah Ravi , Danielson Kirsty , Xiao Chun Yang , Rabideau Dustin , Hanspers Kristina , Murthy Venkatesh , Belcher Arianna , Tackett Michael , Pinzon-Quintero Pablo , Das Avash , Pico Alexander , Januzzi James , Das Saumya
Published: July 2021

Introduction:
Left ventricular reverse remodeling (LVRR) in heart failure (HF) is linked to improved patient outcomes. Current strategies to identify individuals who respond favorably to therapy in HF are limited. Though circulating microRNAs (miRs) show epigenetic regulation of LVRR
in vivo
, there is little clinical data linking plasma-circulating miRs to LVRR. Here we employ a point-of-care microRNA assay, Firefly,
in silico
analyses, and murine models of HF to characterize profiles of circulating miRs that prognosticate LVRR in patients with HF undergoing guideline directed therapy.

Hypothesis:
Profiles of miRs in circulating plasma will prognosticate LVRR in patients with HF better than the current clinical model alone and may play a functional role in LVRR.

Methods:
Plasma from 64 patients from the PROTECT study who had serial echocardiography and available plasma at pre-randomization study visit were run on Abcam’s Firefly platform to assay levels of 51 miRs, selected on prior sequencing efforts (for discovery) and published roles in CVD. miR levels were subject to PC analysis to predict LVRR. Candidate miRs were then validated in a murine model of transverse aortic constriction-induced heart failure (TAC-HF) and their function assessed in cardiomyocyte culture systems.

Results:
Principle component analysis revealed 4 PCs accounting for 62.4% of the observed variation without significant association with extant markers of HF. When PC2 miR levels were added to the clinical model prognostic ability for LVRR improved substantially (AIC 79.5, (OR=6.84, 95% CI 1.81-25.80, P= 0.005). In silico analysis was then applied to generate networks of mRNAs regulated by PC2 miRs. In the murine TAC-HF model, miRs 423, 212, 221, 193b were differentially regulated, as were the predicted targets of these miRs. These miRs, particularly in combination, appeared to be regulators of cardiac hypertrophy
in vitro
.

Conclusions:
Circulating miR profiles in HF improve prognosticative ability for patients who will undergo LVRR over the clinical model alone. Additionally these miRs and their targets are dynamically regulated in murine models of HF, suggesting they may have functional and potential therapeutic utility in addition to their prognostic power.
Abstract 120: The CoExpressed 1a (Liver) Isoform of Carnitine Palmitoyl Transferase 1 is Critical to Cardiac Function and Transcriptional Activation of Metabolic Genes in Aging Hearts
Authors: Carley Andrew , Heydemann Ahlke , Lewandowski E. Douglas
Published: July 2021

Carnitine palmitoyl transferase (CPT) 1 is the rate-limiting enzyme controlling long chain fatty acid (LCFA) oxidation in the heart. The muscle isoform, CPT1b, is predominantly expressed in heart along with the liver isoform, CPT1a, that is co-expressed at comparatively low levels. However, CPT1a content increases in response to pathological stress. Both neonatal and pathologically hypertrophied hearts display elevated CPT1a, despite low LCFA oxidation. To understand the role of cardiac CPT1a, cpt1a
fl/fl
(fl/fl) mice were crossed with mice heterozygous for cre recombinase under the control of the cardiac specific α-MHC promoter to generate cardiac specific cpt1a null mice (cpt1a null). Echocardiography revealed that CPT1a deletion induces age-related declines in systolic function and reduced left ventricular wall thickness. At 5 mos, cpt1a null showed no significant functional defects. At 10 mos, cpt1a null hearts displayed reduced ejection fraction (EF) and fractional shortening (FS) vs fl/fl mice (EF 63±1 fl/fl vs. 21±5 cpt1a null; FS 52±1 fl/fl vs. 18±2 cpt1a null, p<0.0001) with elevated systolic and diastolic volumes. Also at 10 mos, PPARα and PGC1α gene expression declined in CPT1a null hearts by 44% and 22% respectively, vs f/f mice (from arbitrary units referenced to f/f/ control signal, AU). The reduced activation of PPARα and PGC1α genes is the potential consequence of reduced ligand from ATGL-dependent triacylglycerol lipolysis, due to low ATGL gene expression (38% decrease vs. 5 mos f/f, p<0.01, AU). CPT1b gene expression was reduced by 26% (AU) at 10 mos vs f/f/, which is consistent with changes in PPARα, but was not sufficient to alter CPTb protein content. As with CPT1b content, the fractional contribution of exogenous LCFA to oxidative metabolism in hearts perfused with media containing 13C palmitate, glucose, and lactate, was similar among groups, ranging from 0.70-0.73. Therefore, CPT1a does not impact the contribution of LCFA oxidation to mitochondrial energy metabolism in unstressed hearts. However, as hearts age, CPT1a plays a critical role in maintaining transcriptional activation of genes for LCFA metabolism enzymes, via PGC-1α and PPARα and in maintaining normal cardiac function and pathophysiology.
Abstract 170: Differential Regulation of miRNA and mRNA Expression in the Myocardium of Nrf2 Knockout Mice
Authors: Quiles Justin M , Narasimhan Madhusudhanan , Shanmugam Gobinath , Milash Brett , Hoidal John R , Rajasekaran Namakkal Soorappan
Published: July 2021

Background:
Preclinical studies indicate that disruption of the transcription factor nuclear factor, erythroid 2 like 2 (Nrf2) leads to pathological oxidative stress in several tissues. However, the transcriptional role of Nrf2 in the heart, a highly aerobic organ sensitive to redox disturbances and aberrant microRNA (miRNA) activity, remains elusive. Here, we tested the hypothesis that Nrf2 ablation disrupts cardiac miRNA abundance leading to impaired transcriptional regulation of the myocardial defense system.

Methods:
Age-matched wild-type (WT) and Nrf2 knockout (Nrf2
-/-
) mice (n=3-6/group) were used in this study. Independent next-generation sequencing experiments were conducted to evaluate differential expression of mRNA and miRNA in the heart. Real-time qPCR was used to validate RNA sequencing (RNAseq) data.

Results:
RNAseq for mRNA uncovered 152 differentially expressed genes (DEGs) in Nrf2
-/-
hearts, of which 129 were downregulated relative to WT. Novel DEGs previously unreported as Nrf2 targets were detected and Gene Ontology analysis revealed the enrichment (p<0.05) of several biological processes distinct from canonical Nrf2 function including; wound healing, protein folding, cytokine response, cell migration and adhesion. In line with previous reports, altered regulation of apoptosis, stress response and oxidative metabolism was also implicated in Nrf2
-/-
DEG signatures. Next, small RNAseq detected a total of 27 miRNAs (11 up and 16 downregulated) altered in Nrf2
-/-
mice. Validation by qPCR revealed a significant (p<0.05) decrease in miR-10b-5p, miR-674-3p, miR-3535, and miR-378c while miR-30b-5p, miR-208a-5p, miR-350-3p, miR-582-5p, and miR-1249-3p were increased. In silico integration of RNAseq data discovered complementarity between 39 repressed mRNAs and 4 upregulated miRNAs; miR-30b-5p, miR-208a-5p, miR-350-3p, and miR-582-5p. These miRNAs may cooperatively regulate expression in Nrf2
-/-
hearts as 22 DEGs matched with 2 or more miRNAs.

Conclusion:
Our results highlight novel mRNA targets and indicate that Nrf2 regulates a subset of cardiac miRNAs. Biological changes distinct from canonical Nrf2 function suggest that regulatory cross-talk with miRNAs contribute to unique roles for Nrf2 in the myocardium.
Abstract 187: c-Kit Biology Revealed by Two Transgenic Reporter Models.
Authors: Gude Natalie A , Firouzi Fareheh , Nguyen Kristine , Payne Christina , Sacchi Veronica , Alvarez Roberto , Sussman Mark A
Published: July 2021

Background:
The biological significance of c-Kit as a marker of cardiac stem cells, and role(s) of c-Kit+ cells in myocardial development or in response to pathologic injury remain unresolved due to varied findings among investigators and experimental model systems. Alternative experimental models and approaches are needed to achieve a broader perspective of cardiac c-Kit biology that contextualizes discrepant published observations.

Objectives:
Tracking c-Kit expression using transgenesis overcomes limitations inherent to knock-in reporter models. Two novel, inducible transgenic c-Kit reporter models are presented in this study to further elaborate on myocardial c-Kit biology.

Methods:
A previously characterized mouse c-Kit promoter segment was engineered to generate a transgenic mouse in which rtTA transactivator is expressed in c-Kit+ cells (c-KitrtTA). c-KitrtTA crossed to Tet-Responsive-Element(TRE)-Histone2B-EGFP or TRE-Cre lines produces the CKH2B and CKCre double transgenic lines, which express doxycycline-inducible H2BEGFP or Cre proteins in c-Kit+ cells. The CKmTmG triple transgenic mouse, arising from CKCre crossed to the ROSAmTmG reporter line, utilizes doxycycline induced recombination to tag c-Kit+ cells irreversibly with membrane bound EGFP. Endogenous c-Kit and transgenic reporter expression was assessed in adult cardiac myocyte and nonmyocyte cells from these mice under resting and cellular stress conditions using immunohistochemistry and flow cytometry.

Results:
Coincidence of c-Kit and EGFP is observed in approximately 75% of freshly isolated nonmyocyte cells as detected by flow cytometry. A subpopulation of cardiomyocytes express H2BEGFP or mEGFP in the uninjured, doxycycline treated adult heart. H2BEGFP and c-Kit expression increase in myocytes in response to isoproterenol-induced pathologic stress
in vivo
and
in vitro.

Conclusion:
These c-Kit transgenic reporter models provide sensitive, specific, inducible and persistent tracking of c-Kit promoter activation. Results presented here reveal an unexpected role for c-Kit expression in adult cardiomyocytes. Future studies will use both models to investigate c-Kit expression in all cell types during cardiac formation and repair.
Abstract 202: Engineering Artificial Sinus Node by Reprogrammed Cardiomyocytes
Authors: Yufeng Hu , Chen Shih-Ann
Published: July 2021

The lack of clinically relevant sinoatrial node (SAN) disease model makes the pathophysiological investigation and therapeutic development stagnant. We hypothesize that engineering SAN by TBX18 somatic-reprogrammed cardiomyocytes on the three-dimension (3D) scaffold could create an in vitro SAN model, sharing similar features with a native SAN.

Methods:
In addition to neonatal rat ventricular cardiomyocytes (NRVMs) alone, we chose cardiosphere-derived cells (CDCs), or fibroblasts as supportive cells with different mixing ratios to construct engineered SAN. Hydrogel scaffolds including matrigels or platelet gels were used and compared. The engineered tissue was reprogrammed by TBX18 over-expression.

Results:
The over-expression of TBX18 increased HCN4 and CX45 transcriptions in cardiomyocytes. A stable spontaneous beating rate could be created in TBX18-reprogrammed engineered tissue, made of NRVMs and fibroblasts with matrigel scaffold (beating rate, TBX18 vs. control: 105.0 ± 10.7 bpm vs. 35.5±7.1 bpm, n=12, P<0.001). Although spontaneous beating could be observed in reprogrammed engineered tissues by NRVM alone, NRVM with CDCs, or NRVMs with CDCs and fibroblasts, the beating rates were not stable and slower. The beating rate in engineered tissue did not differ between scaffolds of matrigel and platelet gel. However, inter-experimental variation is higher in platelet gels, compared to matrigels. By immunofluorescent staining, an unique spatial distribution of NRVMs and fibroblasts was identified. NRVMs formed the central core of engineered tissues, encapsulated by fibroblasts, which was similar to a native SAN. The application of a sympathomimetic drug (epinephrine) doubled the beating rate of reprogrammed engineered tissue (P=0.02, n=6-8).

Conclusions:
A pilot model of engineered SAN was established by TBX18-reprogrammed cardiomyocytes. The supportive cells such as fibroblasts played an important role in tissue engineering of SAN.
Abstract 204: Protective Roles of Eukaryotic Elongation Factor 2 Kinase on Rat Cardiomyoblast Death Under Glucose-deprived Condition
Authors: Kameshima Satoshi , Okada Muneyoshi , Yamawaki Hideyuki
Published: July 2021

Eukaryotic elongation factor 2 (eEF2) kinase (eEF2K; also known as calmodulin-dependent protein kinase III) has both active and inactive phosphorylation sites. An intracellular energy sensor, AMP-activated protein kinase (AMPK) was reported to activate eEF2K via increasing dephosphorylation at Ser366 (inactive site). Activated eEF2K phosphorylates and inactivates a specific substrate, eEF2, which results in the inhibition of protein translation consuming high energy. Glucose depletion (GD) is one of the primary causes for cardiomyocyte death in the developed cardiac hypertrophy. We have recently found that the expression and dephosphorylation of eEF2K (Ser366) and eEF2 phosphorylation were significantly increased in left ventricle of several cardiac hypertrophy models. However, it is almost unknown whether eEF2K/eEF2 signals affect GD-induced cardiomyocyte death. The aim of this study was to explore it. GD was induced by incubating H9c2 cells in a glucose-free medium. H9c2 cell viability, apoptotic-like nuclear condensation or protein expression was examined using a cell counting assay, DAPI staining or Western blotting, respectively. GD induced H9c2 cell death (p<0.01, n=6) and caspase-3 fragmentation (p<0.05, n=10-12). In addition, GD significantly increased phosphorylation of AMPK (p<0.05, n=6-8) and eEF2 (p<0.01, n=4-8) as well as eEF2K dephosphorylation at Ser366 (p<0.01, n=4-8). eEF2K gene knockdown (eEF2K KD) by siRNA transfection significantly increased GD-induced H9c2 cell death (p<0.05, n=7) and caspase-3 fragmentation (p<0.01, n=9). Moreover, eEF2K KD significantly facilitated GD-induced increase of nuclear condensation (44.0±3.3%, eEF2K siRNA vs. 30.9±2.4%, control siRNA p<0.01, n=5). AMPK KD did not affect GD-induced H9c2 cell death and eEF2K dephosphorylation. In conclusion, we for the first time revealed in H9c2 cells that activated eEF2K might play protective roles in GD-induced apoptosis via the inhibition of caspase-3 fragmentation, whereas AMPK activation is not directly related to the regulation of eEF2K/eEF2 signals in GD condition. The present results suggest eEF2K as a novel pharmacotherapeutic target for cardiac dysfunction.
Abstract 21: Ploidy Alteration of Murine Cardiac Progenitor Cells in Response to Infarction Injury
Authors: Broughton Kathleen M , Wang Bingyan J , Kim Taeyong , Mohsin Sadia , Kubli Dieter , Quijada Pearl , Monsanto Megan , Gude Natalie , Khieu Tiffany , Nguyen Nicky , Rosa Michael , Sussman Mark A
Published: July 2021

Introduction:
Discovery of endogenous cardiac progenitor cells (CPC) prompted intense research efforts in multiple experimental animal models and clinical trials for heart failure treatment. Our lab identified a fundamental difference in ploidy content between rodent (rat, mouse) CPCs possessing mononuclear tetraploid (4n) chromosome content versus large mammal (human, swine) CPCs with mononuclear diploid (2n) content. Ploidy differences raise provocative questions regarding translational applicability of myocardial regeneration in rodents as polyplodization often correlates with enhanced regenerative potential.

Hypothesis:
Mononuclear chromatin duplication in CPCs improves regenerative capacity of the heart through higher stress resistance and overriding senescence cell-cycle arrest.

Methods and Results:
Ploidy of cultured CPCs is consistent and stable ploidy content over increased passages with samples from eight humans, two swine strains, six mouse strains, and seven rat clonal lines as determined by karyotype, confocal microscope and flow cytometry analyses.
In situ
ploidy analysis of CPCs reveals diploid content in human tissue and a mixture of mononuclear diploid and tetraploid nuclei in mouse, confirmed using freshly isolated Lin- c-kit+ CPCs. Tetraploid nuclear phenotype of murine CPCs is markedly different from predominantly diploid (2n) murine c-kit+ cells located in other tissues such as intestine and bone marrow. Higher ploidy content concurrent with expansion of the CPC pool are evident in the border zone at seven days post-infarction in adult FVB mice compared to age and gender matched non-injured hearts.

Conclusion:
Tetraploid c-kit+ cells found within the rodent heart may contribute to species-specific characteristics of stem cells and myocardial regenerative capacity. Future studies will focus upon the biological properties of diploid versus tetraploid CPCs and advantages of polyploid content for mediating myocardial regeneration.
Abstract 235: GJA1-11k Localizes to the Nucleus and Inhibits Cell Cycle Progression
Authors: Epifantseva Irina , Xiao Shaohua , Deng Nan , Hong TingTing , Shaw Robin
Published: July 2021

Gap Junction (GJ) channels, including the most common Connexin 43 (Cx43), are involved in the exchange of ions and small molecules to maintain homeostasis and also have fundamental roles in excitable tissues by facilitating rapid transmission of action potentials between adjacent cells. For instance, synchronization during each heartbeat is regulated by these ion channels at the cardiomyocyte cell-cell border. Numerous studies have found that Cx43 (predominantly the C-domain) is associated with control of cell proliferation, yet it is unclear how Cx43-based membrane channels effect these processes. We recently identified the existence of six endogenous Cx43 isoforms which are produced from the same full-length mRNA molecule in human cells by means of alternative translation and many of the known non-canonical roles of Cx43 can be attributed to these recently identified isoforms. Here we report that the smaller, non-channel related, alternatively translated isoform of Cx43 can directly affect cell growth. We found that 11KDa isoform (GJA1-11k) is localized in the nucleus of HEK293FT cells. GJA1-11k is a more potent growth suppressor than the wild type and full length isoform GJA1-43k. GJA1-11k-mediated growth suppression was achieved by limiting cell cycle progression from G0/G1 to S phase, in part, through regulation of internal translation of GJA1-11k. Nuclear localization experiments indicate that the cell growth-suppressive properties of GJA1-11k is linked to nuclear activity. RNA-seq based genome-wide mRNA transcriptome analysis revealed that the heat shock protein HSPA6, which is normally upregulated in certain cancer cells, was significantly downregulated in GJA1-11k overexpressing cells compared to cells overexpressing the full length isoform, suggesting a potential mechanism and/or new binding protein for the Cx43 C-terminal domain. In general, further understanding of the role of small Cx43 isoforms will provide a way not only to understand the mechanism controlling myocyte proliferation to improve the repair of the myocardium, but also to understand the pathobiology of cell growth associated with tumor suppressive properties of Cx43. It may be possible to develop new therapeutics by regulating alternative translation of Cx43.
Abstract 245: Heart Failure With Preserved Ejection Fraction- a New Animal Model and a Novel Mechanistic Perspective
Authors: Curl Claire L , Danes Vennetia R , Bell James R , Raaijmakers Antonia J , Ip Wendy T , Chandramouli Chanchal , Harding Tristan W , Mellor Kimberley M , Kalman Jonathan , Harrap Stephen B , Delbridge Lea M
Published: July 2021

Diagnosis of heart failure with preserved ejection fraction (HFpEF) is becoming the prevalent form of disease type. Established treatments for heart failure with reduced ejection fraction (HFrEF) have proven minimally effective for HFpEF. This may reflect differences in underlying cardiomyocyte pathophysiology. In HFrEF cardiomyocyte phenotype is characterized by impaired contractile response and diminished systolic Ca
2+
levels. Studies of intact HFpEF-derived cardiomyocytes are lacking. Progress in understanding the etiology of HFpEF has been impeded by limited availability of appropriate pre-clinical models.

Our goal was to validate and characterize a new rodent model of HFpEF, the ‘Hypertrophic Heart Rat’ (HHR), undertaking longitudinal investigations to delineate the associated cardiac and cardiomyocyte pathophysiology.

The selectively inbred HHR strain exhibits adult cardiac enlargement (without hypertension) and premature mortality (40% at age 50 weeks) compared to the control ‘Normal Heart Rat’ (NHR). Echo analyses established that cardiac hypertrophy was characterized
in vivo
by maintained systolic parameters (i.e. ejection fraction at 85-90% control) with marked diastolic dysfunction (i.e. increased E/E’). Diastolic dysfunction was detectable in young adult HHR, as an early disease marker. Histological examination identified regions of focal reparative fibrosis in HHR hearts, most prominent in the transverse midwall area of the left ventricle adjacent to the interventricular septum. Evaluation of cardiomyocyte function using left ventricular myocytes isolated from hearts of 30 week (prefailing ) HHR revealed a hypercontractile phenotype with high Ca
2+
operational levels and arrhythmogenic vulnerability. HHR cardiomyocytes exhibited dramatically increased L-type Ca
2+
channel current density (almost 2-fold), and molecular analyses identified hyperphosphorylation of key sarcoplasmic reticulum Ca
2+
regulatory proteins, without change in total phospho-titin.

These findings strongly support the contention that HFpEF and HFrEF can have different underlying cardiomyocyte phenotypes. New directions for HFpEF therapies are indicated, and the HHR provides a new model for preclinical HFpEF investigations.