bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P2021: Blockade Or Deletion Of The Thromboxane/Prostanoid Receptor Reduces Right Ventricular Stiffness To Maintain Function In Right Ventricular Pressure Overload
Authors: West James D , Colvin Daniel , Moore Christy , Carrier Erica J
Published: November 2022

The increased afterload of pulmonary arterial hypertension (PAH) impairs right ventricular function, as the RV struggles to adapt to increased pressure with remodeling and fibrosis. RV failure is the primary cause of death in PAH, and a frequent cause of death in pulmonary hypertension secondary to pulmonary embolism, emphysema, pulmonary fibrosis, and left-heart failure. There is currently no RV-specific therapeutic to maintain function and prolong patient life. During PAH, cardiomyocytes upregulate cell-surface expression of the G
αq
/IP
3
-coupled thromboxane/prostanoid receptor (TPr), which is activated by multiple endogenous ligands increased in PAH, and leads to calcium influx. TPr activation is also pro-fibrotic in multiple cell types and models. Previous studies have shown reduced fibrosis following TPr antagonism in mouse models of short-term pulmonary hypertension; however this effect has been variable in long-term studies. To more precisely determine the functional effects of TPr activation in RV pressure overload, we used pulmonary arterial banding (PAB) for a direct, sustained increase in pressure, antagonism or deletion of the TPr, and murine cardiac MRI for simultaneous evaluation of the RV and LV. TPr antagonism in a long-term PAB model improved RV ejection fraction and restored LV volume and output by preventing septal bulging and LV eccentricity. This occurred with even short-term antagonism, and was associated with decreased fibrosis in the septum and RV insertion points. Similar results were seen following PAB of universal or cardiomyocyte-specific TPr knockout mice; in all cases, decreasing TPr activation during RV pressure overload reduced RV stiffness even when RV fibrosis remained unchanged. In long-term pressure overload, the improved compliance with antagonism normalized expression of Yap/Taz-associated genes. These results suggest that TPr activation contributes to cardiomyocyte mechanotransduction and deleterious remodeling in response to the RV pressure overload of pulmonary hypertension, and that antagonism of the TPr may preserve RV function to prolong life in PAH patients.
Abstract P2022: Tipifarnib Mediated Protection By Reduction Of Circulating Exosomes In Pressure Overloaded Cardiac Hypertrophy
Authors: Mallaredy Vandana , Roy Rajika , Cheng Zhongjian , Joladarashi Darukeshwara , Magadum Ajit , Gurrala Charan Thej , Cimini Maria , Huang Grace , Garikipati Venkata Naga Srikanth , Wang Chunlin , Truongcao May , Benedict Cindy , Gonzalez Carolina , Koch Walter J , Kishore Raj
Published: November 2022

Hypertrophic cardiomyopathy (HCM) in response to pathophysiological stress is one of the leading causes of heart failure. A growing body of evidence emphasizes the crucial role of exosomes and their modulated content in aggravating cardiac damage due to their inherent intercellular cross-talk abilities during cardiac remodeling. However, the role of circulating exosomes in HCM for the trafficking of pathogenic factors and remodeling the cardiac microenvironment is yet unclear. We investigated the effect of systemic exosome inhibition during cardiac dysfunction in a transverse aortic constriction (TAC) model of heart failure using a recently identified exosome biogenesis inhibitor, Tipifarnib initially developed as Farnesyl transferase inhibitor. In this study, 10-week-old C57BL6J male mice were randomized into three groups i.e., Sham, TAC and Tipifarnib treated (10 mg/kg) TAC. The untreated TAC mice gradually developed hypertrophy and had reduced cardiac functions with a significant increase in heart weight/body weight ratio, cardiomyocyte size and upregulation of hypertrophy and fibrosis associated genes expression by 8 weeks. On the contrary, Tipifarnib treated TAC mice showed remarkably improved cardiac left ventricular functions, reduced cardiac hypertrophy and fibrosis. Notably, Nanosight analysis indicated significantly higher serum exosomes concentration in TAC mice which were substantially suppressed with Tipifarnib treatment. The molecular analysis of the heart tissue revealed Tipifarnib treated TAC mice had reduced expression of the proteins involved in exosome biogenesis in comparison to untreated TAC mice. To gain insight into the cargo of these circulating exosomes, we performed the serum cytokine array and serum exosomes miRNA sequencing in untreated and Tipifarnib treated TAC mice. There was a marked reduction in inflammatory cytokines in serum and differentially expressed exosomal miRNAs with Tipifarnib treatment in comparison to the untreated TAC mice. Overall, our studies suggest the promising potential of Tipifarnib that effectively protects against pressure overload-induced cardiac remodeling and dysfunction by suppressing exosome secretion and altering hypertrophic and fibrotic gene expression.
Abstract P2024: Novel Inhibitor Of P38 Mitogen-activated Protein Kinase Reverses Hypoxia-induced Pulmonary Hypertension In Rats
Authors: Silva Grazielle F , Da Silva Jaqueline S , Silva Marina , Montagnoli Tadeu , Alencar Allan K , Rocha Bruna , Freitas Rosana , Fraga Carlos , Zapata-Sudo Gisele
Published: November 2022

Pulmonary arterial hypertension (PAH) is a disease characterized by pulmonary vascular changes with subsequent right ventricular (RV) failure. This work investigated the hypothesis that a new p38-alpha mitogen-activated protein kinase (p38-alpha MAPK) inhibitor, named LASSBio-1824, could interfere on PH model in rats.

Methods:
HP was induced in male Wistar rats through the exposure to hypoxia (10% O2) during 3 weeks, in combination to i.p. injection of an antagonist of vascular endothelial growth factor receptor (SU5416; 20 mg/kg/weekly). Control rats were kept in normoxia. After 3 weeks, echocardiography images confirmed PH using the pulmonary artery (PA) outflow waveform. After disease onset, animals were randomly divided into three groups: control + vehicle (DMSO), PH + vehicle and PH + LASSBio-1824 (50 mg/kg/day), in which vehicle and LASSBio-1824 were orally administered (gavage) during two weeks. All experiments were in accordance with the Animal Care and Use Committee at Federal University of Rio de Janeiro (039/19).

Results:
Pulmonary acceleration time (PAT; ms) was reduced from 40.0 ± 2.1 (control) to 24.2 ± 1.8 in PH + vehicle group (p < 0.05) and restored to 36.8 ± 2.9 after treatment with LASSBio-1824 (p < 0.05). RV afterload was detected in PH group because of the increase of systolic pressure (mmHg) from 23.2 ± 1.5 (control) to 46.7 ± 2.8 (p < 0.05) which was reduced to 19.4 ± 2.7 with LASSBio1824 treatment. Medial wall thickness (%) of distal pulmonary arteries (PA, < 50 μm) was evaluated by using immunohistochemistry for alpha smooth muscle actin (alpha-SMA) which was increased from 64.0 ± 4.9 (control) to 80.8 ± 2.7 in PH group (p < 0.05), but it was significantly reduced to 55.0 ± 1.3 after treatment with the inhibitor (p < 0.05). Acetylcholine (ACh)-induced maximal relaxation (%) of PA from normal and PH rats, was reduced from 67.5 ± 1.6 to 51.9 ± 4.9 (p < 0.05). Therefore, the treatment with the p38 inhibitor normalized vascular reactivity to 73.0 ± 3.3 %. The immunohistochemistry for c-fos protein, a product of the p38 pathway related to cell proliferation, showed an increase of stained myocyte nuclei (%) in PH animals of 36.5 ± 1.5 when compared to normal group of 20.2 ± 4.2. The inhibitor significantly reduced ratio to 24.0 ± 2.3% (p < 0.05). PH increased the inflammatory condition because the expression of inducible nitric oxide synthase altered from 10.4 ± 1.9% (control) to 31.9 ± 2.2% and it was recovered by the inhibitor to 15.5 ± 2.2% (p < 0.05). In conclusion, the new p38-alpha MAPK inhibitor represents an important approach for the future treatment of PH, since it improves the underlying remodeling and inflammation processes.
Abstract P2025: BET Bromodomain Proteins Are Necessary For TGFβ2-Mediated Myofibroblast Activation
Authors: Antolic Andrew , Jiao Zhe , Alexanian Michael , Ijaz Talha , Padmanabhan Arun , Burke Michael A
Published: November 2022

Fibrosis is a key feature of dilated cardiomyopathy (DCM). Stress signaling triggers the activation of quiescent fibroblasts to myofibroblasts. Bromodomain and extraterminal (BET) epigenetic reader proteins integrate stress-responsive signaling. We explored the role of BETs on distinct inflammatory signaling pathways in cardiac fibroblasts. Fibroblasts were isolated from wild type hearts using the Langendorff procedure. Cell cultures were treated with vehicle, the pan-BET inhibitor JQ1, or siRNA targeting
Smad3
or
Rela
(to inhibit TGFβ and NFκB pathways, respectively) followed by TGFβ2 stimulation. Gene expression was assessed by QPCR, α-smooth muscle actin (αSMA) was quantified by western blot, and NFκB transcription was evaluated using a dual luciferase reporter assay. The interaction between BRD4 and SMAD3 was assessed by coimmunoprecipitation (coIP) in immortalized adult mouse cardiac fibroblast cell cultures treated with TGFβ2, JQ1, or vehicle. TGFβ2-induced fibroblast activation was ablated by JQ1 and
Smad3
siRNA (reduced expression of
Acta2, Col3a1, Postn
and αSMA). CoIP revealed a direct interaction between BRD4 and SMAD3 in fibroblasts, which increased 2.2-fold (p<0.05) after TGFβ2 stimulation. By contrast,
Rela
siRNA alone induced myofibroblast gene expression (
Acta2
, 6.9-fold;
Col3a1
, 7.0-fold;
Postn
, 11.2-fold; p<0.05 vs. vehicle for all), and increased αSMA levels (2.2-fold; p<0.05). This effect of
Rela
siRNA was similar to that observed with TGFβ2 stimulation. Further, TGFβ2 inhibited NFκB-driven gene transcription (-2.9-fold; p<0.05 vs. vehicle). These data demonstrate that BRD4 is a critical co-activator of TGFβ2-SMAD3 signaling and cardiac fibroblast activation. We also found that NFκB signaling is downregulated by TGFβ2, suggesting that NFκB may modulate inflammatory gene network activation.
Abstract P2026: S100a1 Protects Cardiomyocytes From Hypertrophic Growth By Controlling The De-novo Synthesis Of Contractile And Mitochondrial Protein Programs
Authors: Egger Michael , Glaser Manuel , Jungmann Andreas , Syed Azmal A , Krijgsveld Jeroen , Busch Martin , Wade Rebecca , Most Patrick
Published: November 2022

Background and Hypothesis:
Loss of S100A1 expression in heart failure contributes to adverse cardiomyocyte (CM) remodeling via an unknown molecular mechanism. Conversely, the antihypertrophic effect of viral-based restoration of S100A1 expression in diseased hearts is not understood yet. We hypothesized that S100A1 may directly interfere with mRNA translation in CMs.

Methods and Results:
An unbiased mass spectrometry/affinity purification approach using human recombinant S100A1 protein as a bait and murine S100A1
-/-
knock-out CM homogenates as prey identified the translation initiation factors EIF4G, EIF2b, RPS6 and PABP as targets for Ca
2+
-bound (holo) S100A1 protein, amongst other translational regulators. Computational modeling revealed top-scoring binding modes for holo-S100A1’s C-terminus in close proximity to RPS6’s ser-235/236 phosphorylation site harboring domain, and PLA detected S100A1/RPS6 complexes in neonatal ventricular cardiomyocytes (NVCMs). siRNA silencing of S100A1 in NVCMs caused a significant increase in cell size (+44%*) and fetal gene expression (e.g. MYH7 4.2-fold*) with a prompt enhancement of the de-novo protein synthesis rate (+89%*), as assessed by IF, RT-PCR and puromycin incorp. up to 48h (n=3-5, *P<0.05 vs contr). Although phospho-specific IB revealed an increase in RPS6-ser235/236 phosphorylation (+38%, n=10, P<0.05) due to S100A1 knock-down, the activity of the upstream Akt/mTORC2 pathway remained unaltered. A nascent proteome analysis unveiled the significant increase and decrease in the de-novo translation of 554 and 17 proteins (FC>1.5), respectively, in S100A1-silenced NVCMs vs contr. with e.g. MYH7, ALC-1, ACTN2 or Nppb in the top 10 newly synthesized proteins. Heart muscle contraction and metabolism-related GO-terms (e.g. myofibril assembly or FA oxidation and OXPHOS) characterized the top 100 upregulated proteins.

Conclusion:
This is the first study providing evidence for an impact of S100A1 on the mRNA translation machinery in CMs by inhibiting translation of contractile and mitochondrial protein programs required for hypertrophic growth. S100A1 may mechanistically interfere with molecular factors of the translation initiation machinery, which warrants further investigation.
Abstract P2031: Evaluation Of Adenosine Monophosphate-activated Protein Kinase Activator MK-8722 In Human Induced Pluripotent Stem Cell Derived Cardiomyocyte Monolayer And Microtissue (Biowire II) Models
Authors: Imredy John P , Clouse Holly , Lagrutta Armando , Graziano Michael P , Durney Krista M , Hsieh Wilson , Gearhart Jamie , Schriver Brian , Bay Nathan W , Feric Nicole T
Published: November 2022

MK-8722, a systemic pan-AMPK activator improves glucose homeostasis in animal models of diabetes, but causes cardiac hypertrophy
#
. Two- and 3-dimensional tissue (2D,3D) constructs of human induced pluripotent stem cell derived cardiomyocytes (CM) (iCells, Fujifilm/CDI) were used to probe the translatability of cardiac hypertrophy and the role of AMPK in cardiomyocyte function. Chronic 10-day exposure (2 μM and 10 μM MK-8722) of iCell cardiomyocyte 2D monolayers, probed via monitoring of cellular impedance (CardioECR, Agilent), revealed a decrease in beat amplitude (-13%,2μM;-49%,10μM) as well as an increase in excitability, based on the ability to follow higher pacing frequencies (+81%,2uM;+34%,10uM). Direct force measurements in 3D iCell CM tissues (Biowire II, TARA Biosystems) reported loss of contractility when stimulated at 1Hz (-74%,2μM;-90%,10 μM), as well as changes in passive resting tension (+18%,2μM;-75%,10 μM) over chronic six-week exposure. MK-8722 caused a marked increase in Biowire excitability as revealed by a large drop in the voltage threshold of entrainment of paced tissues (-36%,2μM;-43%,10 μM). The increased excitability of both 2D and 3D CM models suggests a cellular correlate of the increased electrocardiographic QRS amplitude observed in rhesus monkeys upon chronic dosing with MK-8722
#
. Hypertrophy of the Biowires could be clearly observed at the end of the 6-week incubation and presented as increased microtissue widths (+5%,2μM; +10%,10μM) and thickened regions of attachment to the chamber force sensors at each end of the Biowire. Volume estimation revealed increases (+22.3%, 2μM; +31.4%, 10 μM) in Biowire tissue volumes that were comparable to heart hypertrophic changes in preclinical test species
#
(mouse, rat, and rhesus monkey). The effect of 10μM MK-8722 was more pronounced when treatment began before tissues had completed a multi-week electrical stimulation maturation protocol, rather than after. The functional effects in both 2D and 3D CM models, and the contractile and structural readouts of the 3D Biowire model were highly informative in the interpretation of the clinical applicability of the pleiotropic cardiac effects of MK-8722 observed in preclinical testing.
#
Myers, et al.
Science
357, p 507-511 (2017)
Abstract P2033: Neddylation Regulates Cardiac Mitochondria Homeostasis
Authors: Zou Jianqiu , Wang Wenjuan , Ayala Juan , Ushio-Fukai Masuko , Fukai Tohru , Yoon Yisang , Chen Weiqin , Li Jie , Su Huabo
Published: November 2022

Neddylation is a reversible post-translational modification that covalently attaches ubiquitin-like protein NEDD8 to target proteins via NEDD8-specific E1-E2-E3 enzymes. Through regulating the function of various cellular proteins, neddylation impacts diverse cellular processes and disease states. We have previously identified an indispensable role for neddylation in cardiac chamber maturation through sustaining YAP signaling in the developing heart. However, the importance of neddylation in post-mitotic hearts remains unclear. Here we report neddylation as a novel post-translation mechanism regulating mitochondrial homeostasis in adult heart. Inducible deletion of NAE1, a regulatory subunit of NEDD8 E1 enzyme, in adult mouse heart resulted in dilated cardiomyopathy, heart failure, and eventually premature lethality. The severe cardiac phenotype was preceded by accumulation of mitophagic vesicles and mitochondrial elongation, accompanied by pronounced upregulation of mitophagic markers and alterations in mitochondrial dynamics proteins. Moreover, we found that neddylation is required for mitochondrial ubiquitination and turnover. Consequently, pharmacological and genetic inhibition of neddylation accumulated damaged mitochondria, impaired mitochondrial membrane potential, inhibited mitochondrial respiration, and increased cardiomyocyte cell death in both neonatal and adult cardiomyocytes. Mechanistically, neddylation of Cullins is essential for the assembly of a functional multi-subunit Cullin-RING ubiquitin ligase, which appears to mediate mitochondrial ubiquitination following mitochondrial damage. Taken together, our findings suggest that neddylation serves as a novel mechanism surveilling mitochondrial quality and is essential for the function of normal adult heart.
Abstract P2034: Photobiomodulation Accelerates Wound Healing In Heart Tissue
Authors: Li Keva , Florio Cristina , Morrell Christopher , Monticone Robert E , Ziman Bruce , Lakatta Edward G
Published: November 2022

Cardiovascular diseases and aging induce stress on the heart, resulting in heart wall thickness and stiffness associated with increased fibrosis. The sinoatrial node (SAN) is the heart’s natural pacemaker composed of a fibrous tissue matrix with a heterogenous cellular population. With aging, activated fibroblasts can create scar tissue, thus commonly resulting in sick sinus syndrome (SSS) and arrhythmias. SSS is produced by idiopathic degeneration of the SAN, requiring implantation of electronic pacemakers. Phototherapy or photobiomodulation (PBM) is a technique with many therapeutic properties that has been applied clinically to treat various forms of tissue pathology. Although there has been evidence that PBM has positive effects on the heart to improve cardiac function and prevent infarction, its age associated changes during wound healing are unknown. This study uses timelapse microscopy and red light (630 nm) or near-infrared (NIR, 810 nm) PBM on SAN support cells (SANspc) isolated from 2 months (2mo) and 2 years (2y) old mice during
in vitro
wound healing. With an average initial gap width of 540 μm, the kinetics and extent of wound closure are measured for each of the SANspc age and treatment groups. Nonirradiated SANspc served as controls. Aging slowed kinetics and increased the extent of wound healing by an average of 1.87 hours earlier than that of the 2mo-SANspc (p < 0.05). Irradiation with red light induces a significantly faster scale of closure for the gap width to close between ½ and ¾ by 0.25 and 0.68 hours, in 2mo- and 2y-SANspc respectively, compared to their corresponding age control. With red irradiation, 2y-SANspc resulted in a significantly faster scale of closure of 0.52 hours compared to 2mo-SANspc. Irradiation with NIR resulted in the opposite effect in both age groups, in which the scale of closure for the wound slowed significantly (0.19 and 0.71 hours in the 2mo- and 2y-SANspc, respectively). In conclusion, these data suggests that SANspc migration and proliferation capacity is faster with aging and red irradiation increases wound closing effect in both age groups. Thus, the enhanced wound healing induced by PBM may be an attractive therapeutic approach to target cardiac fibrosis and promote cardiac remodeling in the aging heart.
Abstract P2036: Investigating Variants Of
Hdac7
In Mammalian Heart Regeneration
Authors: Buddell Tyler , Purdy Alexandra , Peng Xiangwen , Kolell Mary , Swift Samantha , Paddock Samantha , Omeara Caitlin , Patterson Michaela
Published: November 2022

The longstanding belief in the field is the mammalian heart is incapable of regenerating. However, findings regarding the variability of regenerative capacity of mammalian hearts are opening the door for such a possibility. Mononuclear diploid cardiomyocytes (MNDCMs) are a subpopulation of CMs believed to be capable of mounting a regenerative response and the prevalence of the MNDCM population in mammalian hearts is a variable trait. Thus, if we can understand the genetic mechanisms governing the MNDCM variability, it could indicate strategies to stimulate an endogenous regenerative response. Using a genome-wide association approach across a panel of genetically diverse inbred mice, Dr. Patterson identified genes associated with the frequency of MNDCMs in the uninjured adult heart. From this genetic screen, she validated the gene
Tnni3k
as one regulator of CM ploidy and regenerative competence after injury. Here, we follow a second novel locus identified from this screen found on chromosome 15, which includes the gene
Hdac7.
Previously published RNA-seq data on isolated CMs indicate that
Hdac7
expression levels increase during the first postnatal week, a time that coincides with the onset of cellular senescence, CM polyploidization, and the loss of innate regenerative capacity in the heart. Using a CM specific
Hdac7
conditional knockout mouse, we found that loss of
Hdac7
in CMs from birth increases CM DNA synthesis, increases CM ploidy, and increases average CM cell length. Moreover, we identified a protein coding variant of
Hdac7
that corresponds to differences in frequency of MNDCM between members of the mouse diversity panel. Finally, we have gathered preliminary findings that suggest genetic epistasis between
Hdac7
and
Tnni3k.
Specifically,
Hdac7;Tnni3k
double knockout animals display more MNDCMs than
Tnni3k
knockout did on its own. Our findings suggest
Hdac7
negatively regulates DNA synthesis; a key part of the cell cycle that has the potential as a therapeutic target for the stimulation of a proliferation response. In addition, these findings showcase complex genetic interactions regulating CM regeneration, suggesting that combinatorial approaches can enhance regenerative responses.
Abstract P2040: Metolazone As An Adjunct To Loop Diuretics In Acute Heart Failure: A Systematic Review And Meta-analysis
Authors: Shrestha Dhan B , Gaire Suman , Aryal Barun B , Mir Wasey ali Yadullahi , Sedhai Yub Raj
Published: November 2022

Introduction:
Acute heart failure (AHF) affects a significant proportion of the US population. The first-line therapy for decompensated AHF is loop diuretics. However, diuretic resistance in AHF cases is common. In such cases, as add-on therapy, thiazide diuretics are often used to combine two different mechanisms of action. However, the certainty of effectiveness and safety profiles of combining diuretics in cases of diuretic-resistant AHF has not been evaluated in systematic review and meta-analysis considering metolazone, so this study was proposed.

Methods:
The review protocol was registered in PROSPERO (CRD42022302399). PRISMA guideline was followed. Databases were searched using appropriate keywords for eligible papers published before January 5, 2022. The database was screened using the Covidence platform, and analysis was done using Review Manager (RevMan-5.1) software. Odds ratio or mean difference with 95% CI were estimated using fixed or random effect models based on heterogeneity.

Results:
Eight studies among 2999 studies met the inclusion criteria (2 RCTs, six observational studies). The pooled analysis showed no difference in standardized mean difference among the metolazone group and the control group for 24-hours of total urine output (SMD 0.10, CI -0.40 to 0.60) and change in 24-hour 48-hour urine output and 72-hour urine output. There was no difference comparing the addition of metolazone or chlorothiazide to furosemide. However, pooling of the result among those studies comparing furosemide and metolazone with furosemide alone showed a significant increase in the 24-hour total urine output metolazone group (SMD 0.50, CI -0.00 to 0.99). Other outcomes like weight loss, mortality, readmission rate, adverse events did not differ across treatment and control groups.

Conclusion:
Though some benefit in 24-hour urine output was noted on the addition of metolazone to furosemide, our analysis could not demonstrate the benefit of adding metolazone due to lack of adequate studies comparing similar groups in diuretic resistant AHF. Therefore, further RCTs are required in this subject to evaluate the benefit of adding metolazone in diuretic-resistant AHF patients.
Abstract P2050: Acetyl-coa Synthesis Is Required For Chromatin Remodeling In Myofibroblast Differentiation And Persistence
Authors: Lazaropoulos Michael P , Reiter Allison , Gibb Andrew A , Roy Rajika , Elrod John W
Published: November 2022

Heart failure (HF) features fibrotic remodeling by myofibroblasts, a differentiated fibroblast population responsible for wound repair. Discovering mechanisms of myofibroblast formation and function may yield novel targets to delay or reverse HF. Myofibroblasts metabolically reprogram to mediate transcriptional activation of fibrotic genes through chromatin remodeling. We previously reported that α-ketoglutarate production for histone demethylation is essential for cardiac fibrosis, but metabolic regulation of histone acetylation in fibrosis is unclear. ATP-citrate lyase (ACLY) and Acetyl-coenzyme A synthetase 2 (ACSS2) synthesize acetyl-CoA in the cytoplasm and have also been reported to supply substrate for histone acetyltransferases in the nucleus to modify regulatory histone residues. To investigate acetyl-CoA metabolism in myofibroblast formation, we stimulated adult mouse and human cardiac fibroblasts (CFs) with the pro-fibrotic agonist transforming growth factor β (TGFβ) and treated cells with pharmacological inhibitors of ACLY and ACSS2. Either ACLY or ACSS2 inhibition sufficiently reverted differentiated myofibroblasts to a non-fibrotic phenotype, even during continuous TGFβ stimulation. Genetic deletion of ACLY in fibroblasts recapitulated these results. Further, ACLY inactivation was sufficient to decrease global histone acetylation. ACLY deletion, specifically in activated myofibroblasts in the adult mouse heart, slowed LV functional decline and remodeling in a model of pressure overload HF. Our data indicate that ACLY and ACSS2 are necessary for myofibroblast differentiation and persistence. We hypothesize that acetyl-CoA synthesis is necessary for histone acetylation required for activation of myofibroblast genes. Ongoing work will determine the molecular mechanisms of both enzymes in chromatin remodeling and in cardiac injury. In summary, acetyl-CoA producing enzymes are attractive clinical targets to reverse cardiac fibrosis.
Abstract P2052: Lowered Pro-fibrotic And Damage Associated Molecular Pattern Gene Expressions After Exercise Training In A Mouse Model Of Hypertrophic Cardiomyopathy
Authors: Andreassen Kristine , Rixon Chloe , Haugsten Hansen Marie , Hauge-Iversen Ida Marie , Sjaastad Ivar , Christensen Geir , Haugaa Kristina H , Edvardsen Thor , Lunde Ida G , Stokke Mathis K
Published: November 2022

Background:
Hypertrophic cardiomyopathy (HCM) is the most frequent inherited cardiac disease, most often caused by mutations in sarcomere genes. The disease is characterized by left ventricular (LV) hypertrophy, increased risk of arrhythmias and diastolic dysfunction, due to cardiomyocyte hypercontractility, myocyte disarray and fibrosis. Fibrosis is a marker of worse prognosis, adverse remodeling and an increased risk of sudden cardiac death.

Hypothesis:
We hypothesized that exercise training could attenuate fibrosis development in mice genetically predisposed to HCM.

Methods:
We allocated male α-Myh6
R403Q/+
to high intensity interval treadmill running or sedentary behavior (N=11 per group) for a total of 6 weeks. HCM was induced in all mice by cyclosporine A given via the feed for the last 3 of the 6 week exercise protocol. All mice underwent echocardiography and an exercise test at weeks 0, 3, and 6, before harvesting LVs for digital droplet PCR (ddPCR) and a qPCR immune array.

Results:
Exercise training lead to a 2-fold increase in exercise capacity as measured by total distance ran during exercise testing (p<0.0001). In exercised HCM mice, several key fibrosis-related genes were lowered compared to sedentary HCM mice as measured by ddPCR (N=11 per group): Collagen 1 by 51 % (p=0.03), collagen 3 by 54 % (p=0.02), fibronectin by 51 % (p=0.01) and lysyl oxidase by 42 % (p=0.05). An array of 87 selected immune-related genes showed that 39 genes were upregulated >1.5 fold in sedentary HCM mice compared to sedentary wild type controls (N=3 samples, pooled). Twelve upregulated genes belonged to damage associated-molecular patterns (DAMPs). Several of the DAMP genes were lower in exercised HCM mice compared to sedentary HCM mice (N=10-11 per group): Biglycan was lowered by 29 %, versican by 22 % and fibronectin by 55 %. Trends were also found for lowered expression of syndecan 1 (by 26 %), fibromodulin (by 55 %), and tenacin C (by 49 %) (0.07<p<0.08). There was no difference in maximal wall thickness or function measured by echocardiography.

Conclusion:
Exercise training led to a lower expression of key fibrosis-related genes in an inducible mouse model of HCM. Initial results indicate that attenuation of DAMP signaling might be involved in the mechanism for this effect.
Abstract P2053: Role Of Podoplanin Positive Cells Exosomes In Cardiac Inflammation And Amyloidosis
Authors: Cimini Maria , Gonzalez Carolina , Tukel Cagla , Barbe Mary , Lucchese Anna Maria , Wang Chunlin , TRUONGCAO MAY , Huang Grace , Elia Andrea , Mallaredy Vandana , Benedict Cindy , Kishore Raj
Published: November 2022

The extra-cellular-matrix (ECM) composition of scar tissue after myocardial infarction (MI)has been largely investigated. Although fibronectin and collagen are favorable for newmyocyte formation, other components that may increase the scar stiffness and reducethe remodeling of the ischemic area, remain to be identified. Our preliminary studiesidentified primary Serum Amyloid A3 (SAA3) extracellular accumulation that maycontribute to the chronic alteration of the ischemic myocardium’s scar. Specifichistological staining such as thioflavin and Congo red, showed amyloid deposition inmouse hearts 1 month after MI; furthermore, immunohistochemistry for SAA3 detectedthe deposition of the misfolded protein alongside fibronectin and collagen. Serum amyloidamyloidosis (AA) is characterized by deposition of hepatic misfolded protein and SAA3 isthe only amyloid protein that is released locally after inflammation, mostly bymesenchymal progenitor cells. We have reported earlier that two days after MI, a cohortof mesenchymal cells begin to de novo express Podoplanin (PDPN), a plateletaggregation-inducing type I transmembrane glycoprotein, as a signal of activation.PDPN+ cells, in addition to cytokines, release extracellular vesicles including exosomes(Exo) as major paracrine entities driving intercellular communications in homeostasis anddisease. Exo derived from activated PDPN+ cells isolated from MI hearts highly expressSAA3 and injection of activated PDPN+ cell Exo in uninjured healthy mouse hearts leadsto recruitment of immune cells, an extended epicardial fibrosis and amyloidosis with asubsequent impairment in the contractility and increase of the end systolic volumes anddiameters. SAA3 binds Toll-like receptors, and in vitro treatment of bone marrow derivedmonocytes either with PDPN+ cells derived Exo or recombinant SAA3, activated themtowards pro-inflammatory phenotype on contrary these stimuli failed to activate TLR2knocked out monocytes showing an impairment in the expression of major cytokine,chemokine and pro inflammatory markers. Thus, PDPN+ cells in the ischemic heartrelease SAA3 through Exo prolonging inflammation and macrophage recruitment viaTLR2 and contribute to amyloidosis after MI.
Abstract P2059: Poly(rC)-binding Protein-1 (Pcbp1) Is Essential For Heart Development
Authors: Lu Yao Wei , Liang Zhuomin , Guo Haipeng , Fernandes Tiago , Hu Xiaoyun , Espinoza-Lewis Ramon , Cowan Douglas B , Chen Hong , Wang Da-Zhi
Published: November 2022

Rationale:
Proper development of the heart relies on a tightly regulated, yet diverse, pattern of gene expression that is governed by transcriptional, post-transcriptional, and translational processes. Congenital heart disease (CHD) is the most common birth defect and a leading cause of morbidity and mortality in children. While the genetic cause of some types of CHD has been identified, the molecular basis for the rest remains elusive. Poly(rC)-binding protein 1 (Pcbp1) is a RNA-binding protein that regulates RNA processing as well as post-transcriptional and translational processes in a variety of biological systems.

Hypothesis:
Pcbp1 play a critical role in regulating heart development by governing Notch and UPR pathways and mediating proper Aars2 gene splicing.

Methods and Results:
Germline deletion of Pcbp1 results in lethality before embryonic day (E) 8.5. We generated a cardiac-specific deletion of Pcbp1 by crossing Pcbp1-Flox with cTNT-Cre mice (Pcbp1-cKO
cTNT
) and found that Pcbp1-cKO
cTNT
mice is 50% lethal perinatally. Embryonic hearts of Pcbp1-cKO
cTNT
mice displayed ventricular non-compaction and abnormal ventricular apex formation. Deep RNA sequencing of Pcbp1-cKO
cTNT
hearts revealed alteration of gene expression profiles reflective on ventricular maturation delay and dysregulation of Notch and UPR pathways. Interestingly, loss of Pcbp1 in cardiomyocytes disrupts alternative splicing of many important genes including Aars2, a gene associated with congenital mitochondrial cardiomyopathy. Pcbp1 deficiency resulted in creation of an Aars2 exon16-skipping variant, leading to its premature termination. eCLIP-seq showed that Pcbp1 primarily binds to CU-rich motifs at 3’UTR, distal intron and CDS regions of targets, and it interacts with regions of Aars2 transcript near exon 16. Using CRISPR/Cas9 technology, we knocked in loxP sites flanking the exon 16 of Aars2 (Aars2-Flox), and cross the floxed mice with cTNT-Cre mice to generate cardiac-specific exon16 deletion mutant of Aars2 (Aars2-cKO
cTNT
). Intriguingly, abnormality in Aars2-cKO
cTNT
embryonic heart phenocopy aspects of ventricular non-compaction and malformation observed in Pcbp1-cKO
cTNT
heart. Accordingly, the transcriptome from hearts of Pcbp1-cKO
cTNT
and Aars2-cKO
cTNT
display high concordance and similarity and share strikingly common dysregulated pathways.

Conclusions:
We discover a novel function of Pcbp1 in heart development by regulating Notch and UPR pathways. Additionally, Pcbp1 is indispensable for Aars2 gene splicing, whose deficiency is associated with congenital cardiomyopathy. Our findings suggest modulating Pcbp1 in developing hearts may offer a novel therapeutic intervention for congenital heart failure.
Abstract P2061: The Long Non-coding RNA
Schlafenlnc
As A Regulator Of Cardiac Resident Macrophage Function
Authors: Althaus Lara , Heise Kathrin , Esfandyari Dena , Baygun Seren , Theodorakis Evangelos , Gagneur Julien , Meitinger Thomas , Maegdefessel Lars , Wittig Ilka , Bader Michael , Schmidt-Supprian Marc , Dueck Anne , Engelhardt Stefan
Published: November 2022

Introduction:
Cardiac resident macrophages (crMΦs) constitute up to 5% of cells in the murine heart and were shown to play key roles in cardiac homeostasis and disease. Long non-coding RNAs (lncRNAs) are regulatory molecules that impact characteristics such as cell identity, proliferation or migration. However, the function of lncRNAs in crMΦ remains enigmatic.

Objective:
We sought to identify crMΦ-specific lncRNAs and analyze their function
in vitro
and
in vivo
to understand their role during health and cardiac disease.

Methods and Results:
Using RNASeq (>100 million reads/sample) of purified murine crMΦs and single cell Seq of total murine myocardium in health and disease, we could identify the lncRNA
Schlafenlnc
as a highly enriched and abundant lncRNA in crMΦs. Employing the CRISPR-Cas system we successfully deleted the full
Schlafenlnc
locus in a macrophage progenitor cell line. Next, we performed RNASeq of
Schlafenlnc
-/-
macrophages and could observe 2,660 significantly deregulated genes that were enriched in genes associated with chemotaxis and migration. In line with these findings,
Schlafenlnc
-/-
macrophages displayed decreased chemotaxis as well as adhesion using cell-based assays. Furthermore, using RNA-pulldown experiments followed by mass spectrometry analysis and we could identify 27 interaction partners of
Schlafenlnc
, which are involved in processes such as mRNA processing, transcriptional regulation and alternative splicing. Finally, we are currently using cardiac functional measurements, macrophage stainings as well as single cell Seq during health and cardiac disease to analyze the function of
Schlafenlnc in vivo.

Conclusion:
In this study, we could identify the crMΦ-specific lncRNA
Schlafenlnc
as a critical regulator of macrophage migratory functions. Therapeutic targeting of the evolutionary conserved lncRNA
Schlafenlnc
might therefore be beneficial in the treatment of inflammatory cardiac diseases.
Abstract P2063: Circular RNA, circREGEN, Alters Cardiac Regeneration Through Cardiomyocyte Proliferation and Regulation of Oxidative Stress
Authors: Cushman Sarah , Schmidt Arne , Lu Dongchao , Xiao Ke , Huang Cheng-Kai , Abbas Naisam , Thum Thomas , Baer Christian
Published: November 2022

Introduction:
A subtype of non-coding RNA, circular RNAs (circRNAs), formed through backsplicing, are emerging as important regulators of cardiac development and cardiovascular diseases. CircRNAs are known to regulate gene expression through a multitude of functions. Prevalent circRNAs involved in cardiac development and potential regeneration can be determined by focusing on their differential expression in regeneration competent versus non-competent neonatal mouse hearts. We hypothesized that a circRNA that was both highly expressed in day 0.5 tissue and was upregulated under hypoxic conditions (simulating the uterine environment) would have a high potential for regeneration.

Methods and Results:
RNA-seq was performed in day 1 and day 7 mouse hearts and circREGEN was found to be downregulated on day 7 both in RNA-seq analysis and in neonatal mouse tissue samples using RT-PCR. Further circRNA validation was also performed and tissue distribution was measured, showing high expression levels in the heart, specifically, neonatal cardiomyocytes. HL-1 cells, neonatal mouse and rat CMs, neonatal living myocardial tissue, and human induced pluripotent stem cell (iPSC)-derived CMs were subjected to hypoxia (simulating this low oxygen uterine environment) and observed an increase in expression. This data showed that in addition to having high sequence conservation, circREGEN was functionally conserved across species as well. We decided to further examine the expression of circREGEN in living myocardial tissue, an
ex-vivo
model, after cryoinjury and saw an up-regulation. Further experiments were performed to functionally validate circREGEN using siRNAs and overexpression plasmids in neonatal mouse and rat CMs that showed a decrease in proliferation after circREGEN knockdown, and a reduction in ROS after overexpression, showing potential protective and regenerative effects of circREGEN.

Conclusion:
In conclusion, we have promising
in vitro
data, which warrants further functional investigation both
in vitro, ex-vivo,
and
in vivo
, with the ultimate goal to determine if circREGEN has therapeutic potential to regenerate damaged heart tissue after a myocardial infarct by stimulating the proliferation of CMs in the adult heart.
Abstract P2066: Age-related Changes In Posttranscriptional Regulation Of Macrophage VEGF As A Mechanism Of Impaired Inflammatory Arteriogenesis
Authors: Mantsounga Chris S , Sharma Sheila , Lee Cadence , Neverson Jade , Carley Rachel , Choudhary Gaurav , Morrison Alan R
Published: November 2022

Aging and age-related diseases like peripheral arterial disease are associated with impaired inflammatoryarteriogenesis in response to injury. During ischemia, macrophage recruitment and macrophage pro-angiogenic VEGF-A isoform expression contribute to post developmental arteriogenesis. Preliminary datafrom bone marrow-derived macrophages (BMDMs) demonstrated decreased VEGF-A level by both mRNAand protein in aged (52-week-old) and advanced aged (104-week-old) mice when compared to young (12-week-old). Moreover, VEGF-A165a which is VEGF-A proangiogenic splice variant was also reduced inmacrophages from aged and advanced aged mice. The anti-angiogenic VEGF-A165b variant wassignificantly increased in advanced aged mice. We sought to determine the mechanisms which cancontribute to the reduction of pro-angiogenic VEGF-A and consequently reduced inflammatory-mediatedarteriogenesis response in the context of aging. In a hindlimb ischemia model of angio/arteriogenesis, wefound decreased blood flow recovery in aged mice. Moreover, small arterial angiography by microCTconfirmed decreased arteriogenesis. Loss of functional arteriogenesis was associated with decreased muscletissue VEGF-A and VEGF-A165a levels despite adequate macrophage recruitment to the muscle tissue.Moreover, VEGF-A165b was increased in ischemic tissue from advanced aged mice. The mechanism ofreduced macrophage pro-angiogenic VEGF-A165a expression involved decreased mRNA stability withdecreased association of VEGF-A165a mRNA with the RNA-stabilizing protein HuR. In fact, aged BMDMsdemonstrated a loss of HuR binding to VEGF-A165a mRNA with decreased VEGF-A165a mRNA half-life,and consequently decreased inflammatory angio/arteriogenesis.
Abstract P2085: Whole Blood Microfluidics To Assess Direct Oral Anticoagulant (doac) Activity And Reversal In Neonatal Cardiac Patients
Authors: Diacovo Thomas G , Decortin Michael , Diamond Scott L
Published: November 2022

Introduction:
VTEs increase morbidity in neonates with congenital heart disease. Although heparin is the main therapy for prevention / treatment, DOACs are attractive alternatives due to ease of administration, no dependence on antithrombin and wider therapeutic window. Preclinical studies to evaluate their effects on clot formation and antidote reversal are hampered by a lack of low volume assays that permit multiple analyses.

Hypothesis:
A low volume microfluidic system can reproducibly measure activity and reversibility of DOAC-induced alterations in coagulation.

Methods:
A microfluidic device was used to study whole blood (WB) coagulation in response to co-immobilized tissue factor and collagen. Blood was drawn before and 24h after surgery. Platelet and fibrin deposition were monitored by fluorescence microscopy (Apixaban ± andexanet).

Results:
34 neonates were enrolled. Most participants had double ventricle physiology, required bypass and blood products. There was no difference (P>0.05) in Hb / Hct, platelet count, or fibrinogen levels pre- vs post-surgery. Time to clot-induced channel occlusion and platelet / fibrin deposition were similar in post-operative vs adult participants (7.6±3.1 min vs 7.9±1.5 min; Figure 1A), the former prolonged (14.2±1.9 min) and the latter reduced in pre-operative patients. Although Apixaban reduced clot formation (Figure 1B), it prevented channel occlusion in most post-operative neonates vs adults (60% vs 20%). Andexanet reversed its effects, restoring coagulation to non-treatment levels.

Conclusions:
WB microfluidics demonstrated that post-operative neonates are hypercoagulable compared to the pre-operative state and are responsive to apixaban, the effects of which can be reversed with Andexanet.
Abstract P2087: ERBB4-Selective And Sustained Activation By NRG1 Attenuates Atrial Fibrosis And Fibrillation
Authors: Van fraeyenhove Jens , Tubeeckx Michiel , Feyen Eline , Bruyns Tine , De Meyer Guido , Murphy Samuel , Heidbuchel Hein , Segers Vincent , De Keulenaer Gilles
Published: November 2022

Introduction:
Atrial fibrillation (AF) results from electrical and structural remodeling of the atria, in which inflammation and fibrosis play an important role. Current therapy is limited to antiarrhythmic drugs and ablations, but does not target the structural problem. Recent studies showed that neuregulin-1 (NRG1), an epidermal growth factor family member, has anti-fibrotic and anti-inflammatory effects in the myocardium.

Purpose:
To test the effects of JK07, a NRG1 antibody fusion comprising an ERBB3 antagonistic antibody which selectively signals through ERBB4 preferentially over ERBB3, on atrial fibrosis and AF inducibility.

Methods:
Atrial samples were harvested from male rats (Wistar Han, 10 weeks old), cut into small pieces (1-2mm
2
) and kept in low serum medium in the presence or absence of JK07 (5nM).
Col1a1
and
Col3a1
mRNA was quantified after 24-72 hours. AF inducibility was tested in a first AF model in which male mice (C57BL/6N, 12-15 weeks old) were treated with angiotensin-II (Ang-II, 4 weeks, osmotic mini-pumps, 3000 ng/kg/min), and in a second AF model in which mice were fed with a high fat diet (HFD, 8 weeks, 60% Kcal fat) inducing severe weight gain (56±3% increase compared to 23±4% with regular chow). In both models, AF inducibility was tested by 5 runs of programmed electrical stimulation (PES) with a trans-jugular octapolar catheter. AF inducibility (% mice inducible by ≥3 PES-runs) and duration of PES-induced AF (AF duration) were recorded. Mice were randomized for treatment with vehicle or JK07 (2x/week, 1mg/kg, IV, n=5-7/group).

Results:
In cultured atrial samples,
Col1a1
and
Col3a1
mRNA expression gradually increased up to 2-3 fold over 3 days. JK07 robustly attenuated this effect by 59±17% (p<0.05). In mice, both Ang-II and HFD significantly increased AF inducibility and AF duration. In Ang-II mice, JK07 attenuated AF inducibility (from 57% to 20%) and AF duration (from 33.3 ± 15.1 to 1.5 ± 1s). In HFD mice, JK07 significantly attenuated AF inducibility (from 57% to 0%) and AF duration (from 10.9±3.2s to 0.76±0.5s, p<0.05).

Conclusions:
These results show anti-fibrotic effects by selective ERBB4 stimulation with JK07 in atrial tissue
in vitro,
together with AF-preventive effects in two unrelated mouse models.