bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 280: The Effect of YAP/TAZ on Glycolysis and Mitochondrial Oxidative Phosphorylation in Cardiomyocytes
Authors: Kashihara Toshihide , Sadoshima Junichi
Published: October 2019

The heart requires a high rate of adenosine triphosphate (ATP) production to maintain healthy cardiac function and viability. Anaerobic glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) are the main metabolic pathways by which ATP is generated in mammalian cells, including cardiomyocytes. Yes-associated protein 1 (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ), which are major downstream effectors of the Hippo signaling pathway, play an important role in the regulation of heart size and cellular homeostasis of cardiomyocytes. However, interplay between YAP/TAZ and cardiac energy metabolism has been poorly understood. Here we examined the effect of YAP/TAZ on glycolysis and OXPHOS in isolated neonatal rat ventricular cardiomyocytes (NRVMs). Extracellular acidification rate (ECAR; an index of glycolysis) and oxygen consumption rate (OCR; an index of OXPHOS) of NRVMs transduced with adenoviruses harboring YAP, TAZ, or LacZ (control) for 6 days were assessed by Seahorse XFe96 analyzer. Both YAP and TAZ significantly increased not only the ECAR of basal glycolysis (1.5-fold and 1.3-fold, respectively, p<0.05), glycolytic maximal capacity (1.4-fold and 1.3-fold, respectively, p<0.05), and glycolytic reserve capacity (1.3-fold and 1.2-fold, respectively, P<0.05) (n=12-15) but also basal OCR (1.3-fold and 1.3-fold, respectively, p<0.05) (n=6-10) compared to control LacZ. Quantitative PCR analysis showed that mRNA levels of Pfkm, a key glycolytic enzyme, and subunits of protein complexes I though V in the electron transport chain are significantly increased (1.3-1.9-fold, n=5-6, p<0.05) by both YAP and TAZ compared with control. These results suggest that YAP/TAZ increase ATP production by accelerating glycolysis and OXPHOS in cardiomyocytes.
Abstract 287: Altered Mitochondrial Flash Activity and mPTP Opening in the Aged Heart Are Reversed by Elamipretide Treatment
Authors: Zhang Huiliang , Szeto Hazel , Alder Nathan , Rabinovitch Peter S
Published: October 2019

Rationale:
The mitochondrial theory of aging pinpoints mitochondria as the major reactive oxygen species (ROS) production site and target of oxidative stress during aging. Mitochondrial flash (mitoflash) is recently discovered excitable event that is a manifestation of ROS production combined with pH change and coupled with mitochondrial permeability transition pore (mPTP) opening. Our preliminary data shown that 8-week SS-31 peptide (elamipretide) protects the functional performance of the aged mouse heart. This study aims to investigate mitoflash activity and mPTP opening in the aged heart, especially whether and how SS-31 protects the heart function involving modulation of these activities.

Methods and Results:
By using confocal microscopy imaging on the isolated rat cardiomyocytes with overexpression of mitochondrial targeted cpYFP, we found mitoflash activity in the cells from old (26 mo) was higher than that in young (5 mo) rat cells (2.2 ± 0.2 in old vs 1.3 ± 0.2 in young, /1000μm
2
/100s, n=27-64, p<0.05). SS-31 10 μM treatment normalized the mitoflash activity to the young level. Similarly, by photon-excitation induced mPTP opening, we found that aging increased the mPTP opening and 1 μM SS-31 stabilized the mPTP. Furthermore, by using Seahorse Assay on intact primary isolated cardiomyocytes, we found mitochondrial basal respiration in cells from old mice was higher than that in young mouse cells, however, the maximal respiration rate was without significant difference. The increased basal respiration was attributed to a higher proton leak in old cardiomyocytes (131 ± 14 in 24 month vs 94 ± 11 in young, pmol/min/800cells, n=17-35, p<0.05). Interestingly, acute (2 hrs)
in vitro
treatment of the old cardiomyocytes with SS-31 100 nm decreased proton leak. Moreover, SS-31 (100 nM) decreased the superoxide production as measured by ratio of MitoSOX Red to MitoTrackerGreen.

Conclusion:
These results indicate that SS-31 directly protects cardiac aging through rapid rejuvenation of mitochondrial respiration in cardiomyocytes, and in particular, by reducing proton leak, mitochondrial flash activity, and decreasing the mPTP opening. This study helps to uncover the mechanism of the cardiac aging and the protective effect from SS-31 treatment.
Abstract 295: Cysteine 202 of Cyclophilin D is a Site of Multiple Post-translational Modifications and Plays a Role in Cardioprotection
Authors: Amanakis Georgios , Sun Junhui , Brody Matthew , Bauer Tyler , McGinty Shane , Boylston Jennifer , Liu Chengyu , Molkentin Jeffery , Murphy Elizabeth
Published: October 2019

Cyclophilin-D (CypD) is a well-known regulator of the mitochondrial permeability transition pore (PTP), the main effector of cardiac ischemia/reperfusion (I/R) injury characterized by oxidative stress and calcium overload. However, the binding of CypD to the PTP is poorly understood. Cysteine 202 of CypD (C202) is highly conserved across species and can undergo redox-sensitive post-translational modifications. To study the importance of C202, we developed a knock-in mouse model using CRISPR where CypD-C202 was mutated to a serine (C202S). In a Langendorff perfused heart model, infarct size is reduced by 51% in CypD-C202S hearts compared to WT (n=4, p=0.003). Isolated cardiac mitochondria from CypD-C202S mice also have a 49% higher calcium retention capacity compared to WT (n=5, p=0.011). We hypothesized that oxidation of C202 might target CypD to the PTP and that the cardioprotection in CypD-C202S mice is due to a post-translational modification on C202. Indeed, isolated cardiac mitochondria subjected to oxidative stress exhibit less binding of CypD-C202S to the proposed PTP component ATP synthase by 48% compared to WT (p=0.012, n=4). We previously found C202 to be S-nitrosylated in ischemic preconditioning. Cysteines can also undergo S-acylation (attachment of a fatty acid), and C202 matched a S-acylation motif. To assess S-acylation of CypD-C202 we employed a resin-assisted capture. Lysates were treated with N-ethylmaleimide to block free cysteines, and then with hydroxylamine to free cysteines from fatty acids. Subsequently, free cysteines were captured by a sepharose resin, followed by western-blot of the eluate. CypD-C202 is abundantly S-acylated, but CypD-C202S hearts exhibit less S-acylation than WT by 63% (n=6, p<0.001). We investigated the importance of S-acylation of CypD-C202 in cell death in a mouse embryonic fibroblast (MEFs) model. CypD-C202S MEFs exhibit 20% less oxidative cell death than WT (n=5, p=0.003). Inhibition of S-acylation with 2-bromopalmitate attenuates oxidative cell death in the WT by 20% (n=5, p=0.018), while CypD-C202S MEFs are unaffected. Thus, S-acylation and oxidation of CypD-C202 possibly target CypD to the PTP, sensitizing it under oxidative stress and making them potent regulators of cardiac I/R injury.
Abstract 296: B7-33, a Functionally Selective Relaxin Receptor 1 Agonist, Exerts Protective Effects Against Myocardial ischemia-Reperfusion Injury in Mice
Authors: Devarakonda Teja , Mauro Adolfo G , Guzman Geronimo , Cain Chad , Das Anindita , Praveen Praveen , Hossain M A , Salloum Fadi N
Published: October 2019

Background:
B7-33 is a peptide derived from the B-chain of human relaxin, and is shown to elicit biased signaling at relaxin receptor 1 (RXFP1) in human cell lines. The influence of B7-33 on myocardial ischemia-reperfusion (IR) injury and associated cardiac adverse remodeling is unknown.

Methods and Results:
Primary cardiomyocytes and cardiac fibroblasts were isolated from adult CD1 mice and subjected to simulated ischemia-reoxygenation (SIRO). Myocytes were placed in 1% O
2
chamber for 40 min, followed by 1 h of reoxygenation with control (myocyte media) or B7-33 (10, 50 or 100 nM)-infused media. Trypan blue staining showed a significant decrease in cell death with B7-33 concentrations of 50 nM and 100 nM (
Fig. A
).Treating myocytes with B7-33 for 15 min exhibited a dose-dependent increase in Erk1/2 phosphorylation, which reached statistical significance at 100 nM (
Fig. B
). Fibroblasts subjected to 4 h of hypoxia and 15 h of reoxygenation had increased viability (MTT assay) with B7-33 at all concentrations (
Fig. C
).
In vivo
, CD1 mice were subjected to IR injury via coronary artery ligation for 30 min, followed by 24 h of reperfusion. Vehicle (saline) or B7-33 (10 μg/kg) was injected i.p. at the onset of reperfusion. After 24 h, B7-33-treated mice demonstrated decreased infarct size (TTC stain) (
Fig. D
) and preserved fractional shortening (M-mode echo,
Fig. E
) compared to vehicle-treated mice.

Conclusion:
Reperfusion therapy with B7-33 reduces infarct size post-MI, preserves cardiac function and protects cardiomyocytes and fibroblasts against SIRO. We propose that B7-33 might be effective against acute MI and a possible alternative to recombinant relaxin for clinical utility.
Abstract 298: Downregulation of Delta and Kappa Opioid Receptors in Post-heart Transplantation in Human and Rats
Authors: Gebrie Mebratu A , Rungatscher Alessio , Linardi Daniele , Andrioli Anna , Ahmed Sajeela , Ahmed Naseer , Ismail Mahmoud , Forni Alberto , Tessari Maddalena , San Biagio Livio , Soave Alessandro , Milani Elizabetta , Innamorati Giulio , Luciani Giovanni B , Faggian Giuseppe
Published: October 2019

Over the past decade, multiple data regarding local opioid regulation of heart function have been demonstrated. However, opioid receptors (ORs) have not yet characterized in post-heart transplantation. Since a transplanted heart is devoid of innervations and autonomic innervations modulate ORs, we assessed the hypothesis that a possible down-regulation of k-OR and δ-OR in heart transplants.

Endomyocardial biopsies were collected in 15 male patients at 30 days of orthotopic heart transplantation and in 15 control male patients during aortic valve surgery in human. Another study performed in 20 heterotopic heart transplants (HHT) and 20 naïve hearts of Sprague-Dawley male rats. The mRNA transcript encoding the Oprd1 and Oprk1, KOR and DOR proteins, distribution, and cellular localization were identified using RT-qPCR, Western blot, IHC, and IF, respectively.

Transcripts of mRNA encoding the Oprd1 and Oprk1 were detected in hearts of human and rats. Expression of Oprd1 (p=0.036) and Oprk1 (p = 0.004) was significantly reduced in transplanted hearts. In Western blot analysis, KOR and DOR levels of expression were significantly lowered (p=0.03, KOR; p=0.01, DOR) in HHT compared to naïve hearts in rats. Immunohistochemistry analysis demonstrated a lighter labeling on the distribution of DOR and KOR in heart transplants` tissue in both rats and human. In both species, dual labeling myocardial isoforms (KOR and DOR) co-localized in cardiomyocytes of transplant and control hearts.

These findings suggest that the down-regulation of DOR and KOR receptors in hearts transplant of both human and rats might have a possible increased susceptibility to ischemia-reperfusion injury.
Abstract 299: Engineering Rodent TRPV1 to Mimic Chicken TRPV1 Reduces Capsaicin-induced Calcium Influx in H9C2 Cells
Authors: He Shufang , Sinharoy Pritam , Gross Eric Richard
Published: October 2019

Background and Aim:
Birds, unlike mammals, are resistant to the spicy hot sensation when consuming chili peppers. The inability for birds to sense the hot sensation is accredited to genetic divergence in the transient receptor potential vanilloid 1 (TRPV1) when compared to mammals. Recently, we described how TRPV1 is important for regulating myocardial ischemia-reperfusion injury in rodents. Here, we tested our hypothesis that K710 is a crucial mediator of capsaicin-induced calcium influx in myocardial H9C2 cells.

Methods and Results:
Sequence alignment of the C-terminus TRP domain of TRPV1 among species was performed. Unlike mammals having K710 TRPV1, birds instead have N710 TRPV1 (Fig 1A). Site-directed mutagenesis of K710N was performed on wild type (WT) rTRPV1 with a pCMV6-entry vector. The empty vector, WT and K710N plasmids were transfected into H9C2 cells and the cellular localization of TRPV1 channel were detected by immunofluorescent staining and channel activity by capsaicin challenge with live cell calcium imaging with Fura-2AM. Interestingly, the K710N mutation caused the TRPV1 channel to be retained intracellularly and co-localized with PDI, an endoplasmic reticulum marker (Fig 1B, Bar=25μM). K701N cells treated with 1μM capsaicin showed a reduced response to capsaicin compared to wild type TRPV1 transfected cells (Fig 1C-E, 011±0.01 in WT
vs
. 0.03±0.01 in K710N, n=10, P<0.01).

Conclusions:
Site-directed mutagenesis of K710N caused rTRPV1 to be insensitive to capsaicin and suggests a potential target for limiting calcium influx from myocardial ischemia-reperfusion injury which could be a novel protective strategy to prevent damage from a heart attack.
Abstract 304: Pgc1a Activation by Pterostilbene Ameliorates Acute Doxorubicin Cardiotoxicity via Reducing Mitochondrial Oxidative Stress Through Enhancing Ampk and Sirt1 Cascades
Authors: Liu Dong , Qiao Shubin , Yuan Jiansong
Published: October 2019

Background:
Doxorubicin (DOX) is a widely used and potent anticancer agent, but DOX dose-dependently induced cardiotoxicity greatly limits its use in clinic. Pterostilbene, a natural analog of resveratrol, is a known antioxidant and exerts myocardial protection. The present study explored the actions and detailed mechanisms of pterostilbene on DOX-treated cardiomyocytes.

Experimental Approach:
We investigated the effects of pterostilbene on established acute DOX-induced cardiotoxicity models in both H9c2 cells treated with 1 μM DOX and C57BL/6 mice with DOX (20 mg/kg cumulative dose) exposure. Cell viability, ATP content, oxidative parameters, transmission electron microscopy, western blot analyses were respective performed. AMPK and SIRT1 siRNA, Compound C and EX527 treatment were further conducted to elucidate the underlying mechanism about the involvement of AMPK, SIRT1 and PGC1α cascades.

Key Results:
Pterostilbene markedly upregulated the cell viability and ATP content in DOX-treated H9c2 cells. Both
in vitro
and
in vivo
studies revealed that pterostilbene inhibited the acute DOX exposure-caused oxidative stress and mitochondrial morphological disorder via the PGC1α upregulation through activating AMPK and via PGC1α deacetylation through enhancing SIRT1. However, these effects were partially reversed by knockdown of AMPK or SIRT1
in vitro
and treatment of Compound C or EX527
in vivo
.

Conclusion and Implications:
Our results indicate that pterostilbene protects cardiomyocytes from acute DOX exposure-induced oxidative stress and mitochondrial damage via PGC1α upregulation and deacetylation through activating AMPK and SIRT1 cascades.
Abstract 307: Chemogenetic Activation of Paraventricular Oxytocin Neurons Reduces Cardiac Dysfunction During Heart Failure
Authors: Rodriguez Gonzalez Jeannette , Dyavanapalli Jhansi , Rocha Carla , Dwyer Mary Kate , Schloen John , Schunke Kathryn J , Mendelowitz David M , Kay Matthew W
Published: October 2019

There is a distinctive cardiac autonomic imbalance in HF consisting of increased sympathetic activity and decreased parasympathetic tone. Recent work has indicated that activation of paraventricular nucleus (PVN) oxytocin neurons increases co-release of oxytocin with glutamate to elevate cardiac vagal tone yet little is known regarding the long-term (16 week) benefit. We hypothesize that activation of oxytocin neurons in the PVN will blunt the progression of HF by reducing inflammation and fibrosis at 16 weeks post-HF initiation than in untreated counterparts. Rats underwent trans-ascending aortic constriction (TAC) to induce HF. In a subset of HF rats (TAC+OXT), oxytocin neurons were activated by excitatory designer receptors exclusively activated by designer drugs (DREADDs). Rats were implanted with ECG telemetry devices and underwent a treadmill protocol to measure peak effort capacity and heart rate recovery (HRR). Cardiac structural measurements were obtained using echocardiography. After 16 weeks, hearts were rapidly excised, Langendorff perfused to measure mechanical function, and prepared for histology and western blot analysis. Indices of cardiac function (stroke volume, ejection fraction, cardiac output, and fractional shortening) indicated preserved function in TAC+OXT compared to TAC. HRR after peak effort capacity values were lowest in TAC and were significantly greater in TAC+OXT (p≤0.05), indicating preservation of cardiac parasympathetic tone. LV developed pressure was significantly impaired in TAC compared to sham as well as significantly improved in TAC+OXT compared to TAC (p≤0.05). Contractility and relaxation were significantly impaired in TAC compared to sham (p≤0.05), while TAC+OXT animals had values not different from sham. TAC animals had collagen III levels 5X higher than TAC+OXT. Histological analysis indicated increased fibrosis in disease groups compared to sham. TAC+OXT had IL-1β levels significantly lower than TAC (p≤0.05). PVN oxytocin neuron activation slows the decline of cardiac function in 16 week post- TAC rats, likely by reinstating cardioprotective parasympathetic activity. This novel method has translational potential since intranasal oxytocin administration has been used in the clinic.
Abstract 321: RBFOX2 is critical for maintaining alternative polyadenylation patterns in cardiomyoblasts
Authors: Cao Jun , Belanger KarryAnne , Jaworski Ela , Rayavara Kempaiah , Nutter Curtis , Ji Ping , Elrod Nathan , Verma Sunil , Widen Steven , Wagner Eric J , Garg Nisha , Routh Andrew L , Kuyumcu-Martinez Muge
Published: October 2019

Background:
The RNA binding protein RBFOX2 is implicated in human heart diseases. However, RBFOX2-regulated RNA networks are not well defined. RBFOX2 has a well-characterized role in alternative splicing (AS) while accumulating evidence suggests that RBFOX2 may also have a role in alternative polyadenylation (APA). Recent studies showed that RBFOX2 binds to regions close to poly(A) sites in the 3’UTR of pre-mRNAs. In addition, RBFOX2 binds to the cleavage and polyadenylation specificity factors. Therefore, we aimed to determine whether RBFOX2 has a role in regulating APA.

Method:
We employed poly(A)click sequencing (PAC-seq) and DPAC (Differential Poly(A) Cluster analysis) computational pipeline to identify differential poly(A) usage and mRNA abundance. We also used nanopore sequencing to identify different spliced variants and the coordinated AS and APA events.

Results:
We report that knockdown of RBFOX2 in embryonic rat heart derived cells leads to altered alternative polyadenylation (APA) of hundreds of genes. RBFOX2-mediated APA changes impacted both mRNA levels and generation of different gene isoforms. Nanopore sequencing identified full-length transcripts regulated by RBFOX2 and revealed RBFOX2-mediated isoform switches via both APA and AS in cardiac cells. Notably, RBFOX2-regulated APA networks affect genes such as
Tpm1
and
Tnnt1
involved in cardiac contractility. Identification of RBFOX2-regulated RNA networks provides novel insights into the pathogenesis of heart diseases in which RBFOX2 is involved and pave the way for designing therapeutics.

Conclusions:
RBFOX2 regulates alternative polyadenylation via splicing dependent and independent mechanisms. RBFOX2-mediated APA affects mRNA levels of contractile genes.
Abstract 323: A Novel Human Specific Long Noncoding RNA PG1 Controls Human Cardiogenesis via Regulating Beta-catenin Activity
Authors: Han Lei , Li Yang , Yang Lei
Published: October 2019

Long noncoding RNAs (lncRNAs) can regulate gene expression, signaling pathway, cellular and tissue functions at various stages. Recently, accumulating evidences suggest that lncRNAs play important roles in cell fate specification and organ development. We found hundreds of lncRNAs, which show lineage-specific expressions in human embryonic stem (ES) cell-derived multipotential cardiovascular progenitor cells (MCPs), cardiomyocytes (CMs), smooth muscle cells (SMs) or endothelial cells (ECs), by using whole transcriptome sequencing. Among MCP-specific lncRNAs, we identified a novel human lncRNA, PG1, which controls cardiovascular differentiation from human pluripotent stem cells. We found PG1 could regulate the early segregation of nascent mesoderm cells towards cardiac verse hematopoietic fate. Knockout of PG1 in human induced pluripotent stem (iPS) cells and H1 ES cells by CRISPR/Cas9 significantly increased hematopoietic differentiation from pluripotent stem cells when compared with wild type controls. Furthermore, our data suggest PG1 could bind beta-catenin protein to modulate Wnt signaling activity to regulate nascent mesoderm specification. In summary, we identified a novel human specific lncRNA, PG1, which plays an essential role in early stage human heart development.
Abstract 324: Translation Determines the Acute Cardiac Response to Ischemia/Reperfusion
Authors: Hofmann Christoph , Younesi Fereshteh S , Schwerdt Ole M , Bioleau Etienne , Katus Hugo A , Dieterich Christoph , Völkers Mirko
Published: October 2019

Introduction:
Since transcription is a relatively slow process, translation of preexisting mRNA networks might affect cardiac gene expression thereby rapidly adapting the myocardium to stress before changes in transcription take place. The mTOR kinase links nutrient and energy levels to protein synthesis by regulation of mRNA translation. Inhibition of mTOR complex 1, the responsible pathway for translational regulation, protects against reperfusion injury, however the underlying mechanisms are unknown. Through rapid restoration of myocardial energy levels upon reperfusion an mTOR-dependent translatome could adapt early changes in gene expression inducing a maladaptive gene network, independent from transcription, which mediates reperfusion injury.

Hypothesis:
Reperfusion induces an mTOR-mediated overshooting protein synthesis response that contributes to reperfusion injury by rapidly and selectively translating preexisting mRNAs.

Methods and Results:
To induce reperfusion injury mice were subjected to 60 minutes of LAD ligation and increasing times of reperfusion. Puromycin incorporation showed an overshooting protein synthesis response of cardiac myocytes upon reperfusion that was especially prevalent in the peri-infarct region. Protein synthesis was induced by increased formation of the eIF4F translation initiation complex through the mTORC1-4EBP1 signaling axis. Cardiac myocyte specific ribosome profiling combined with RNA-seq of hearts 2 days after reperfusion showed that the acute cardiac response to reperfusion was mediated by translation of specific mRNAs encoding proteins which determine early events of reperfusion such as leukocyte infiltration and inflammation. Inhibition of the mTORC1-4EBP1-eIF4F axis attenuated this specific protein synthesis response. Importantly, 4EGI-1, a specific inhibitor of eIF4F complex formation, bypassed off-target effects of previously used mTOR inhibitors and protected cardiac myocytes from cell death upon reperfusion.

Conclusions:
During reperfusion protein synthesis is strongly induced in the peri-infarct region by mTORC1 and contributes to reperfusion injury by rapidly translating specific mRNAs that mediate the early gene expression response to reperfusion.
Abstract 326: FTO-mediated mRNA Demethylation Regulates Cardiac Contractile Protein Expression and Function
Authors: Mathiyalagan Prabhu , Adamiak Marta , Mayourian Joshua , Sassi Yassine , Liang Yaxuan , Agarwal Neha , Jha Divya , Zhang Shihong , Kohlbrenner Erik , Yin Xiaoke , Chen Jiqiu , Trivieri Maria G , Bouchareb Rihab , Singh Rajvir , Fish Kenneth , Ishikawa Kiyotake , Mayr Manuel , Lebeche Djamel , Hajjar Roger J , Sahoo Susmita
Published: October 2019

Background:
Exciting new discoveries in RNA biology underscore the importance of post-transcriptional chemical modifications to mRNAs (epitranscriptome) in regulating RNA stability, nuclear export, cellular compartmentalization, splicing, translation and degradation. The most abundant and functionally relevant modification in RNA, N6-methyladenosine (m6A) is reversibly demethylated by one of the m6A demethylases, fat mass and obesity-associated protein (FTO) whose function in the mammalian heart remains incompletely understood.

Materials and Methods:
We used clinical human samples, preclinical pig and mouse models and primary cardiomyocytes to study m6A and FTO in the heart and in cardiomyocytes. We modulated FTO expression using AAV9 (in vivo), adenovirus (in vivo and in vitro) and siRNAs (in vitro). We investigated m6A-induced changes to contractile protein expression using m6A RNA immunoprecipitation sequencing (MeRIP-seq) and stable isotope labeling of amino acids in cell culture (SILAC).

Results:
We discovered in human heart failure that reduced FTO expression is associated with aberrant increase in m6A mRNA methylation, which is conserved in swine and mouse models of myocardial ischemia (MI). AAV9-mediated FTO gene delivery in mouse MI attenuated m6A increase and improved cardiac function with enhanced contractility, angiogenesis and reduced fibrosis. At the molecular level, FTO-mediated mRNA demethylation serves to increase contractile protein expression in mouse hearts as well as in isolated primary cardiomyocytes. By comparing human and mouse transcriptome-wide m6A maps with SILAC proteomic profiling from cardiomyocytes, we identified FTO-mediated m6A demethylation is transcript-specific and leads to altered protein expression of several key contractile, angiogenic and regenerative proteins.

Conclusion:
Using new RNA-based investigations, we uncovered a novel regulatory layer beyond the genome working at the level of epitranscriptome governing cardiac function. Our findings on the dynamic nature of the cardiac m6A-epitranscriptome will lead to deeper understanding of the mechanism of cardiac remodeling on one hand and innovative therapeutic interventions on the other.
Abstract 354: Golgi Localized β
1
-adrenergic Receptors Stimulate Golgi PI4P Hydrolysis by PLCε to Regulate Cardiac Hypertrophy
Authors: Wei Wenhui , Nash Craig A , Irannejad Roshanak , Smrcka Alan V
Published: October 2019

Phospholipase C (PLC)-mediated PI4P hydrolysis pathway at the Golgi is important for regulating cardiac hypertrophy. cAMP stimulates this pathway via Epac-mediated activation of Rap1 which directly binds to and activates PLCε. Here, we demonstrate that a membrane permeant β-adrenergic agonist, dobutamine (dob), and the endogenous β-adrenergic agonist, norepinephrine (NE), induce Golgi PI4P hydrolysis in neonatal rat ventricular myocytes (NRVMs) and adult ventricular myocytes. However, a membrane impermeant β-adrenergic agonist, isoproterenol (iso), does not. In addition, a membrane permeant βAR antagonist, metoprolol, but not a membrane impermeant antagonist, sotalol, fully reverses dob-mediated PI4P hydrolysis. Taken together, this suggests that internal β-ARs are required for inducing PI4P hydrolysis. Subsequently,we used YFP-tagged mini Gs proteins that recruit to Gs-coupled receptors upon their activation, to monitor the location of activated receptors. Dob and NE induced robust and rapid recruitment of mini Gs to the plasma membrane and the Golgi, however, iso only induces recruitment to the plasma membrane. Additionally, inhibition of PLCεat the Golgi with either siRNA or the RA1 domain of PLCε, or the use of an Epac inhibitor also inhibits dob-mediated PI4P hydrolysis. This suggests that PI4P hydrolysis by dob requires Golgi-localized PLCεand Epac.Inhibition of the βAR with Golgi-targeted Nb80 prevented PI4P hydrolysis by both dob and NE confirming that Golgi-localized βARs are required for PI4P hydrolysis.Also, Oct3 transporter inhibitors prevent NE-induced PI4P hydrolysis but dyngo, an inhibitor of receptor internalization, had no effect. This indicates that agonist transport and not receptor internalization is required for NE-mediated Golgi βAR activation and PI4P hydrolysis. Furthermore, dob stimulates an increase in cell size and ANF expression in NRVMs that is significantly inhibited by metoprolol but less effectively by sotalol. Taken together, these data suggest that Golgi βARs are involved in mediating cardiomyocyte hypertrophy and may serve as a novel target for treating heart failure.
Abstract 361: Deacetylation of Lc3 Drives Autophagy and Proteome Remodeling in Skeletal Muscle During Oncometabolic Stress
Authors: Karlstaedt Anja , Vitrac Heidi , Gould Benjamin D , Salazar Rebecca , Taylor David , Spivia Weston , Soetkamp Daniel , Dinh An , Raedschelders Koen , Burks Helen , Schiffer Walter , McNavish Daniel , Hanson Blake , Gottlieb Roberta A , VanEyk Jennifer , Taegtmeyer Heinrich
Published: October 2019

Metabolic rewiring is a hallmark of cancer and muscle cells. In isocitrate dehydrogenase 1 and 2 mutant tumors, increased plasma levels of the oncometabolite D-2-hydroxyglutarate (D2-HG) are associated with systemic effects, including myopathy. Our recent
in vivo
work showed that increased D2-HG supply by IDH-mutant cells causes heart and skeletal muscle atrophy, and decreases cellular ATP and NADH. Although heart failure and cachexia in cancer are commonly associated with chemotherapy, cancer survivors have a 5-fold increased risk of heart failure independent of any cytostatic treatment. The connection between metabolic and proteomic remodeling in this context remain poorly understood. We hypothesize that D2-HG-mediated alpha-ketoglutarate dehydrogenase (AKGDH) inhibition in myocytes results in metabolomic perturbations, increases autophagy and proteomic remodeling. Here, we report that LC3, a key regulator of autophagy, is activated in the nucleus of myocytes in presence of D2-HG through deacetylation by the nuclear deacetylase Sirt1. Activation of Sirt1 is driven by increased NAD
+
levels through D2-HG-mediated AKGDH inhibition. We used LC3 mutants with arginine and glutamine replacements at lysine residues to show that deacetylation of LC3 at K49 and K51 by Sirt1 shifts LC3 distribution from the nucleus into the cytosol, where it is able to undergo lipidation at pre-autophagic membranes. Live cell imaging with GFP-tagged LC3 in L6 myocytes indicated that the cycle of acetylation-deacetylation allows LC3 to redistribute from the nucleus to the cytosol within less than 24 h. Co-immunoprecipitation of LC3 followed by proteomics analysis revealed that LC3 binds to dynein in presence of D2-HG. Furthermore, D2-HG promoted skeletal muscle atrophy and reduced grip strength in wild-type C57BL/J6 mice in vivo. Using LC-MS/MS-based proteomics and metabolomics combined with RNA-sequencing, we assessed the effect of D2-HG on a systems level in skeletal muscle. Pathway-enrichment analysis revealed that D2-HG induces upregulation of key metabolic enzymes involved in glycolysis and the pentose phosphate pathway. In short, autophagy activation supports proteome remodeling in muscle cells during IDH-mutant leukemia.
Abstract 371: Chromatin Remodeling Mechanisms by Bromodomain PHD Finger Transcription Factor in Cardiac Hypertrophy and Heart Failure
Authors: Awad Salma M , Zahid Esra , Al-Shalan Sarah , Alaiya Ayodele , Al-Tinawi Qamar , Al-Sheikh Amar , Kunhi Muhammed , Al-Haffar Kamar , Al-Buraiki Jehad , Eyjolsson Atli , Al-Habib Waleed , Poizat Coralie I
Published: October 2019

Alteration of chromatin conformation has a profound effect on gene expression, and can lead to heart failure, which remains a major cause of death worldwide. ATP-dependent chromatin remodelers utilize the energy of ATP to remodel chromatin in specific regions of the genome. However, the mechanisms of ATP-dependent chromatin remodeling and how they connect to the genome remain poorly understood. We have discovered that the nucleosome remodeler Bromodomain PHD-finger Transcription Factor (BPTF), the largest subunit of the Nucleosome Remodeling Factor (NURF), plays an important role in cardiac hypertrophy. Our preliminary data show that BPTF is enriched in failing human hearts, a phenomenon similarly observed in mouse hearts subjected to pressure overload hypertrophy (TAC). BPTF is up-regulated in primary neonatal rat ventricular myocytes (NRVM) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) stimulated with the hypertrophic agonist phenylephrine (PE). Strikingly, specific silencing of BPTF in NRVM and hiPSC-CMs, inhibits the expression of the atrial natriuretic peptide (ANP), a marker of cardiac hypertrophy. Chromatin immunoprecipitation performed in NRVM show that BPTF binds to the ANP promoter, but not to GAPDH. Proteomics experiments performed in failing human hearts revealed a multi-protein complex formed between BPTF and novel chromatin regulators. At the level of the nucleosome, BPTF in combination with one of its partners, remodels chromatin by facilitating nucleosomes “sliding” along the DNA. This mechanism is mediated by the binding of BPTF to histone H3 trimethylated at Lysine-4 (H3K4me3), an “active” mark of transcription also up-regulated in the early phase of cardiac hypertrophy in NRVM and in mouse TAC hearts. Our study suggests a novel chromatin signature controlled by BPTF, that dictates specific gene patterns implicated in pathological cardiac hypertrophy. Our study uncovers a new epigenetic mechanism controlling pathological cardiac remodeling and heart failure.
Abstract 372: CRISPR-based Gene Activation for Transcriptional Reprograming of Mammalian Cardiomyocytes
Authors: Schoger Eric , Carroll Kelli J , McAnally John , Tan Wei , Liaw Norman , Iyer Lavanya M , Noack Claudia , Zimmermann Wolfram H , Bassel-Duby Rhonda , Zelarayan Laura C
Published: October 2019

Cardiac remodelling is accompanied by silencing of genes necessary for cardiac homeostasis including Krüppel-like factor 15 (
Klf15
), an anti-hypertrophic factor. Due to a lack of tools for gene activation, we aim to adapt a CRISPR-based approach for gene expression control for mammalian cardiomyocytes. We generated a transgenic mouse model (TG) expressing an enzymatically inactive, gRNA-programmable Cas9 protein under
Myh6
promoter control fused to transcriptional activators (VPR) to drive gene expression. As a proof of concept, we systemically injected mice at postnatal day P4 with AAV9 carrying a triple gRNA expression cassette containing gRNAs targeted to the
Klf15
promoter region. Significant
Klf15
activation was observed at P10 up to 10-fold compared to controls in three independent TG lines. This
Klf15
activation was sufficient to drive target gene expression of
Aldh2
and
Adhfe1
(controls = WT + saline, WT + AAV9, TG + saline, n ≥ 3 per group, ANOVA and Bonferroni correction). Importantly, at the neonatal stage, the
Klf15
promoter is characterized by low H3K27ac presence indicating that endogenous gene activation can enhance transcription from an epigenetically inactive locus. We demonstrated that CRISPR/Cas9 mediated loss of
KLF15
expression results in impaired contractile function in a 3D model of engineering heart muscle, similar to the
Klf15
-/- mouse heart phenotype. Encouraged by the evolutionary conserved
KLF15
-mediated mechanisms, we aimed to generate a human induced pluripotent stem cells, which constitutively express dCas9VPR (hIPSC-dCas9VPR) for gene activation in hIPSC-derived cardiomyocytes to complement the
in vivo
findings upon disease. We targeted the transgene integration to the AAVS1 site by CRISPR gene editing, confirmed correct integration by PCR followed by sequencing and hIPSC-dCas9VPR were successfully differentiated into beating cardiomyocytes. To this end, we have tested gRNAs targeted to the
KLF15
promoter region in HEK293 cells and observed significant
KLF15
activation of up to 5.5-fold (n = 3, ANOVA and Bonferroni correction). In summary, we report transgenic mammalian tools for endogenous gene (re-) activation purposes to identify potential therapeutic targets for preventing heart failure progression.
Abstract 373: Regulation of Hypoxia by Chromatin Reader Protein Kinase C Binding Protein 1 (PRKCBP1)
Authors: Schunke Kathryn J , Shohet Ralph V
Published: October 2019

PRKCBP1 (also known as RACK7 and Zmynd8) is a polyvalent chromatin reader known to cooperatively bind acetylated and methylated nucleosomes. Recently it has been shown to regulate transcription and cancer progression by coordinating histone methylation modifications affecting enhancer and promoter regions of genes. The role of PRKCBP1 in the cardiac myocardium is unexplored. Hypoxia-inducible factor 1α (HIF-1α) upregulation and stabilization is a common feature of both cancer and myocardial ischemia, promoting cellular functions such as proliferation, glucose metabolism and angiogenesis. Here we investigated the mechanism by which PRKCBP1 modulates the cardiac hypoxic response.

We hypothesize that PRKCBP1 inhibits the HIF-1 response in the hypoxic heart by reducing enhancer activity of HIF-1 target genes and altering availability of HIF-1 binding sites.

We have found that in transgenic mice with a mutation that increases HIF-induced expression of PRKCBP, the effect of induced oxygen-stable HIF is markedly diminished. These mice did not exhibit the typical HIF-1 over-expression phenotype of dilated vessels, increased heart size and reduced ventricular function. Semi-quantitative rtPCR analysis of mouse neonatal cardiomyocytes transfected with CMV-driven expression plasmids for PRKCBP1 and oxygen-stable HIF-1α showed striking reduction of multiple HIF-1 target genes such as PDK1 (45% reduction relative to Ehbp1) compared to the HIF-1α plasmid alone. RNAi mediated knockdown of PRKCBP1 removed this negative regulation (65% increase). Analysis of human PRKCBP1 and HIF-1α ChIP-seq data indicate that PRKCBP1 binds to the enhancer of 78% of HIF-1 regulated genes. ATAC-seq data suggest that PRKCBP1 affects genome-wide chromatin accessibility, with loci-specific modifications at numerous HIF-1 target genes, such as EGLN3. These data suggest that PRKCBP1 may be acting both by modulating enhancer activity
in cis
- to HIF-1 target genes and by preventing HIF-1 binding to hypoxia response elements of target genes.

We have discovered a new regulator of HIF-1 action that modifies the hypoxic response, likely through chromatin remodeling. This new form of regulation may modify the pathophysiology of ischemia and provide new targets for therapy.
Abstract 407: Modeling Atrial Fibrillation in a Dish Using Atrial iPSC Derived Cardiomyocytes
Authors: HONG Liang , Zhang Meihong , Youn Seock-Won , Lambers Erin , Sridhar Arvind , Menon Ambili , Chalazan Brandon , Wu Joseph C , Rehman Jalees , Darbar Dawood
Published: October 2019

Mutations in multiple genes have been linked with familial atrial fibrillation (AF) but the underlying pathophysiologic mechanisms and implications for therapy remain poorly understood. To characterize the electrophysiological phenotype of an AF-linked
SCN5A
mutation and assess novel mechanism-based therapies, we generated patient-specific atrial induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a carrier with an SCN5A-E428K mutation and an unaffected family member using a differentiation protocol optimized for atrial differentiation generated atrial iPSC-CMs. The atrial iPSC-CMs carrying SCN5A-E428K displayed increased window and late Na+ current (INa,L), increased beating frequency and irregularity with triggered beats and prolongation of the action potential duration (APD) versus control atrial iPSC-CMs. The multi-electrode array (MEA) recordings of mutant atrial iPSC-CMs showed spontaneous arrhythmogenic activity with beat-to-beat irregularity. We further showed that targeted inhibition of the INa,L with ranolazine normalized the aberrant electrophysiologic phenotype in the SCN5A-E428K atrial iPSC-CMs and mexiletine reversed the sodium channel gating defects, and reduced beating frequency and irregularity. Our study illustrates the potential use of atrial iPSC-CMs for modeling AF in a dish, elucidating the underlying cellular mechanisms, and identifying novel mechanism-based therapies custom-tailored for individual patients.
Abstract 413: Adipose-derived Stem Cells With Thymosin Beta 4 Enhanced Neovascularization in Mouse Ischemic Hind Limb Model
Authors: Kim Jong-ho , Lim I-Rang , Joo Hyung Joon , Hong Soon Jun
Published: October 2019

Adipose-derived stem cells (ASCs) have potential of differentiating into endothelial lineage and are easily obtainable, therefore they are promising candidates for stem cell therapy. Thymosin beta 4 (Tb4) is G-actin sequestering protein that exerts a broad range of functions such as cell migration and angiogenesis. In this study, we investigated the effects of Tb4 on angiogenic and endothelial differentiation potential of ASCs. Exogenous treatment of Tb4 (100 ng/mL) induced a significant increase in endogenous mRNA expression of Tb4, and morphological changes with increased cell length in ASCs. In addition, Tb4-treated ASCs showed significantly higher endothelial differentiation potential than untreated ASCs after the induction of endothelial differentiation for 3 weeks. The expression of endothelial markers such as PDGFRb and CD144 were significantly enhanced by the treatment of Tb4 with the endothelial differentiation induction media compared to untreated ASCs. Indeed, Tb4-treated ASCs expressed higher expression of angiogenic genes including Ang-1, vWF, Tie1, CXCR4, uPAR, FGFR4, IGFR2, and VE-Cadherin than untreated ASCs, both cultured in the endothelial differentiation induction media. Moreover, a scratch wound healing assay revealed that the treatment of Tb4 significantly increased cell migration compared to untreated and Tb4-siRNA transfected ASCs. In the microbead sprouting assay, the treatment of Tb4 significantly augmented the number of sprouts per bead and the length of sprouts compared to untreated and Tb4-siRNA transfected ASCs. Futhermore, transplantation of ASCs with Tb4 induced neovascularization improved the blood flow in mouse ischemic hind limb models, compared to sham, ASC-transplanted, and Tb4-transplanted groups. Taken together, therapeutic application of ASCs with Tb4 could be useful to enhance endothelial differentiation and neovascularization.
Abstract 420: S-nitrosylation Promotes Cell Cycle, Cell Viability and Proliferation by Activating the Snail/Slug Pathway in miPSC-derived CM
Authors: Salerno Alessandro , Wanschel Amarylis , Hatzistergos Konstantinos , Dulce Raul , Balkan Wayne , Hare Joshua
Published: October 2019

Introduction:
Epithelial-to-mesenchymal transition (EMT), is a crucial step in cardiac differentiation. Snail and Slug are increased and E-cadherin is decreased during EMT. Expression of
Snail
and
Slug
is reduced by GSK3β, a key regulator of the Wnt/β-catenin signaling pathway. S-nitrosylation (S-NO) of GSK3β inhibits its activity.

Objective:
We hypothesized that in the absence of S-nitrosoglutathione reductase (GSNOR), the Snail/Slug pathway is activated in miPSC-derived cardiomyocytes.

Methods:
We compared the initial growth and differentiation of iPSCs derived from GSNOR
–/–
(iPSC
GSNOR-/-
) and wild type (WT [control], iPSC
WT
) mice on days (D) 0-6 in cell culture. Cell morphology was assessed and MTT, BrdU and transwell invasion assays performed to determine cell proliferation, invasion and migration. EMT-related transcription factors and members of the GSK3β pathway were measured at D0-D6.

Results:
During early differentiation, iPSC
GSNOR-/-
cells exhibited accelerated loss of pluripotency markers and, from D4, greater proliferation/differentiation, and apparent EMT compared to iPSC
WT
. MTT, BrdU and migration assays demonstrated that loss of GSNOR stimulated cell proliferation and migration.
Slug
and
Snail
were upregulated and
E-Cadherin
was downregulated in iPSC
GSNOR-/-
suggesting that increased NO levels reduced
GSK3β
activity in iPSC
GSNOR-/-
.

Conclusions:
Our results suggest that the deletion of GSNOR affects early CM differentiation and promote EMT. These findings have important implications for regenerative medicine and provide new targets for iPSC-based therapy.
Abstract 421: Organ-on-chip Model for Investigating Autonomic Innervation of the Cardiac Microenvironment
Authors: Soucy Jonathan R , Torregrosa Tess , Hosic Sanjin , Arteaga Sebastian M , Koppes Abigail N , Koppes Ryan A
Published: October 2019

Cardiac sympathovagal imbalance is a dysfunction of the parasympathetic and sympathetic nervous systems (PSNS and SNS) that, in tandem, regulate cardiac output in response to environmental stimuli. This autonomic nervous system (ANS) imbalance increases the risk of cardiac arrhythmias and sudden cardiac failure due to an inability to modulate heart rate following overexertion or excessive stress. Therefore, there is an immediate need to understand the underlying cellular mechanisms of cardiac innervation to develop new strategies to restore ANS balance. Moving away from the complexity and variability of
in vivo
models, we developed an
in vitro
microphysiological model of the cardiac ANS. Custom “cut and assembled” microfluidic organ chips were fabricated to support the 3D culture of cardiac cells and ANS neurons within a biomimetic scaffold. Using this system, on-chip innervation of the cardiac microenvironment was confirmed via immunostaining (Figure 1). Further, we demonstrated trending differences in cardiomyocyte beating between those innervated by each ANS neuron population (PSNS vs. SNS: 37.4 ± 6.7 bpm vs. 44.8 ± 7.9 bpm, n = 8, p = 0.0716) via video microscopy and custom software (MATLAB®). Investigations to alter cardiomyocyte function to promote the innervation of different neural populations within the cardiac microenvironment are ongoing. Specifically, beat rate will be increased/decreased with pharmacological agents and innervation quantified using commercial neuron tracing software (Neurolucida®). Altogether, the development and use of this model will enable the systematic investigation of novel therapies to restore cardiac sympathovagal balance.
Abstract 430: Impaired Autophagic Off-rate Causes Cardiac Aging in Progeria Mouse Model
Authors: Bando Yasuko K , Kamihara Takahiro , Murohara Toyoaki
Published: October 2019

Background/Introduction:
Aging is known to be one of the primary causes of heart failure. Werner syndrome is one of the aging disorder due to dysfunction of DNA helicase-regulatory protein (WRN) and exhibiting progressive phenotype of natural aging. Ample evidences demonstrated progeria may promotes autophagic disorder. [PURPOSE] To elucidate whether autophagic disorder may be mechanistically responsible for cardiac aging.

Methods:
We employed progeria mouse model harboring amino acid (AA) substitution of WRN at position 577 (WRN-K577M, 18 week-old male).

Results:
WRN-K577M exhibited diffuse left-ventricular (LV) hypertrophy, enhanced fibrosis, and diastolic LV dysfunction with preserved systolic ejection fraction. DNA microarray analysis of WRN-K577M heart revealed that 253 genes were upregulated compared to age- and gender-matched wild-type counterpart. Sixteen genes were increased > 4 fold higher than wild-type as follows: hypertrophy (Myh7, Klkb11), fibrosis (CTGF), inflammatorymolecules (Ap1s3, Pla2g2e, Has1, MMP9), and oxidative stress (catalase). Cardiac aging markers (PARP-1, p53 and γH2AX) increased in heart of WRN-K577M with concomitant increase in oxidative stress (DHE staining) and apoptosis (TUNEL). Notably, autophagic turnover markers (p62 and LC3-II/I) were increased in myocardium of WRN-K577M, which was refractory to fasting-induced increase in on-rate of autophagy. Consistently, one of the key regulators of autophagy is the target of rapamycin, TOR kinase, which is the major inhibitory signal that shuts off autophagy with concomitant activation of Akt signaling. Collectively, these data indicates that the on-rate step of autophagy could be pathologically augmented with disruption of downstream process, i.e. impaired off-rate pathway that modulates turnover of autophagosome and lysosome under cardiac aging observed in WRN-K577M. Changes in autophagosomal and lysosomal accumulation were monitored by specific fluorescence indicators and chloroquine (50 microg/g body weight) augmented lysosomal accumulation with reduced LC3-II/I ratio.

Conclusion(s):
In WRN-mutant progeria model, off-rate disorder of cardiac autophagy is, at least in part, the cause of cardiac aging.