bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract EC103: A Novel Nuclear Role For REDD1 In Cardiac Insulin Sensitivity And Resistance
Authors: Wheeler Mason , Ibetti jessica , Koch Walter J , Pfleger Jessica M
Published: November 2022

Insulin resistance (IR) is a hallmark of type II diabetes (TIID) and causes cardiac dysfunction independent of hypertension and coronary artery disease, or diabetic cardiomyopathy (DC). Impaired metabolism and energetics are a major cause of DC, however the molecular mechanisms underlying these defects remain ill-defined. Our unbiased RNA sequencing (RNA-Seq) studies identified ‘regulated in development and DNA damage (REDD)1’ as a potential critical regulator of cardiac insulin sensitivity, as a 1.5-fold increase in REDD1 expression was observed in the hearts of mice treated with acute insulin (1 hour (h)) versus vehicle (n=3). REDD1 is an insulin-sensitive, negative regulator of mTORC1 and global REDD1 deletion leads to whole body IR. Interestingly, cardiac REDD1 is also upregulated in several models of IR. Thus, we hypothesized that while REDD1 is critical for cardiac insulin sensitivity, sustained elevated levels contribute to IR via chronic inhibition of mTORC1. Our findings confirm that acute insulin stimulation or chronic high fat diet (HFD) (2 to 16 weeks) induce cardiac REDD1 expression
in vivo
(1.7-fold±0.1, n=6). In neonatal rat ventricular myocytes (NRVM) or Hap1 cells, insulin (1 h) or palmitate (24 h) also induce REDD1 expression (1.3-fold±0.1, n=3 or 1.6-fold±0.1, n=9, respectively), which indeed inhibits mTORC1. Notably, insulin treatment following HFD or palmitate, does not further enhance REDD1. In addition, subcellular fractionation shows that REDD1 is uniquely detected in nuclear and chromatin fractions and increases with insulin (1.5-fold±0.1, n=6). We confirmed REDD1 nuclear localization via microscopy and chromatin binding via chromatin immunoprecipitation-deep sequencing (ChIP-Seq) (n=5, pooled). In addition, we determined that REDD1 primarily binds the transcription start sites of metabolic genes and increases with insulin, corresponding to reduced gene expression. Overall, our findings suggest that insulin-inducible cardiac REDD1 exists as part of negative feedback loops that prevent the overactivation of mTORC1 and metabolic gene expression, contributing to insulin sensitivity. Further, sustained fat-induced REDD1 expression, may drive IR via chronic inhibition of mTORC1 and metabolic gene expression.
Abstract GS109: Ponatinib-induced Cardiotoxicity Is Driven By S100A8/A9-NLRP3-IL1β Signaling Mediated Excessive Inflammation
Authors: Sultan Tousif , Singh Anand P , Umbarkar Prachi , Galindo Cristi , Wheeler Nicholas , zhang qinkun , Prabhu Sumanth D , Lal Hind
Published: November 2022

Background:
The tyrosine kinase inhibitor (TKI) ponatinib is the oral drug for the treatment of chronic myelogenous leukemia (CML) patients with T315I (gatekeeper) mutation. Pharmacovigilance analysis of FDA and WHO datasets have revealed that ponatinib is the most cardiotoxic agent among all FDA-approved TKIs in a real-world scenario. However, the mechanism of ponatinib-induced cardiotoxicity is unknown. The cardiotoxic effects of ponatinib remained hidden in preclinical studies and were only revealed in phase 2 clinical studies and by the subsequent meta-analysis. The lack of well-optimized mouse models has hampered the
in vivo
cardio-oncology studies. To this end, here we aimed to investigate the hidden cardiotoxic mechanism of ponatinib and strategies to prevent the cardiotoxic manifestations.

Methods:
We employed wild-type C57BL/6, cardiovascular (CV) comorbidity models e.g., transverse aortic constriction (TAC)-pressure overload (cardiac comorbidity) and high-fat diet-fed ApoE
-/-
(vascular comorbidity), to investigate the cardiotoxic mechanism of ponatinib. Echocardiography was performed to assess cardiac function. Comprehensive immune profiling was performed to identify ponatinib-induced immune activation using flow cytometry analysis and cytokine assay.

Results:
Echocardiographic assessment of ponatinib treated high-fat diet-fed ApoE
-/-
and pressure overload (PO) murine model showed a significant decline in cardiac function, suggesting the key role of CV-comorbidities in ponatinib-induced cardiomyopathy. A combination of multiple
in vitro
and
in vivo
models was employed to delineate the underlying molecular mechanisms. An unbiased RNA-Seq analysis identified the enrichment of dysregulated inflammatory genes, including a multi-fold upregulation of alarmins S100A8/A9 as a top hit in ponatinib-treated hearts. Mechanistically, we demonstrate that ponatinib activates the S100A8/9-TLR4-NLRP3-IL-1β signaling pathway in cardiac and systemic myeloid cells (monocytes and neutrophils),
in vitro
and
in vivo
, thereby leading to excessive myocardial and systemic inflammation. Excessive inflammation was central to the cardiac pathology because interventions with broad-spectrum immunosuppressive glucocorticoid dexamethasone or direct NLRP3 inhibitor Cy-09 nearly abolished the ponatinib-induced cardiac dysfunction. Taken together, these findings uncover a novel mechanism of ponatinib-induced cardiac inflammation leading to cardiac dysfunction.

Conclusions:
These findings uncover a novel mechanism of ponatinib-induced cardiac inflammation leading to cardiac dysfunction. Our results provide critical preclinical data and rationale for a clinical investigation into immunosuppressive interventions to mitigate ponatinib-induced cardiotoxicity.
Abstract GS114: Maladaptive Myeloid Lipid Metabolism Promotes Cardiac Diastolic Dysfunction Through Splenic Hematopoiesis
Authors: Filipp Mallory , Ge Zhi-Dong , Shah Sanjiv J , Thorp Edward B
Published: November 2022

Rationale:
Diastolic dysfunction, including during heart failure with preserved ejection fraction (HFpEF), is a widespread morbidity with limited therapeutic options. Though heterogeneous in etiology, major risk factors include hyperlipidemia, hypertension, and elevated inflammation. Inflammatory macrophages have been implicated in the promotion of diastolic dysfunction; however underlying mechanistic insight remains in its infancy.

Objective:
We set out to define the heterogeneity of the cardiac immune response during diastolic dysfunction to identify cell and molecular protagonists relevant to HFpEF. We hypothesized that systemic metabolic perturbations during HFpEF were promoting cardiac inflammation and fibrosis.

Methods and Results:
Diastolic dysfunction was induced by a combination of high fat diet (HFD) and iNOS inhibition (L-NAME). scRNAseq of the myocardium revealed significant alterations in the composition of cardiac macrophage subsets. Gene ontology analyses depicted a specific induction of pro-fibrotic and lipid handling pathways, including the fatty acid receptor Cd36. In live animals, myeloid deficiency of Cd36 prevented diastolic dysfunction and was associated with dampened cardiac inflammation. When investigating immune cell sourcing during HFpEF, it was found that a splenic hematopoietic niche formed in wild-type mice. Within splenic macrophages, HFpEF-associated risk factors activated mitochondrial metabolism, as well as expression of vascular cell adhesion molecule-1 (VCAM-1), the latter which has been shown to retain hematopoietic stem cells in the spleen. In vitro treatment of splenic macrophages with high fat was sufficient to induce expression of Vcam1, an effect found to be mediated by mitochondrial reactive oxygen species activation of the transcription factor Spic.

Conclusions:
Our data suggest a critical role for myeloid metabolism in the sourcing of detrimental-monocyte-derived macrophages from the spleen, to the heart.
Abstract P1006: Cross-lineage Potential Of Ascl1 Uncovered By Comparing Diverse Reprogramming Regulatomes
Authors: Wang Haofei , Keepers Benjamin , Liu Jiandong , Qian Li
Published: November 2022

Direct reprogramming has revolutionized the fields of stem cell biology and regenerative medicine, making it possible to interconvert somatic cell fates without intermediary pluripotency. Although studies have identified different transcription factor (TF) cocktails that can reprogram fibroblasts into different cell types, the common mechanisms governing how reprogramming cells undergo transcriptome and epigenome remodeling (i.e.,
regulatome
remodeling) have not been investigated. Here, by characterizing early changes in the regulatome of three different types of direct reprogramming - induced neurons, induced hepatocytes, and induced cardiomyocytes - we identify lineage-specific features as well as common regulatory transcription factors that act in a context-dependent manner for each type of reprogramming. Of particular interest, we discover that the neuronal factor Ascl1 possesses cross-lineage potential; together with Mef2c, it navigates reprogramming fibroblasts through a targeted and potent iCM trajectory. Single-cell multi-omics reveal that A+M reprogramming terminates in a more mature iCM phenotype than MGT. Finally, through ChIP-seq and RNA-seq, we find that Mef2c drives the shift in Ascl1 binding away from neuronal genes towards cardiac genes, guiding their co-operative epigenetic and transcription activities. Given the relatively short DNA coding sequence of Ascl1 (696bp) and Mef2c (1299bp), our novel A+M reprogramming cocktail has the potential to be delivered within a single AAV vector. Such a strategy is not feasible with classic cocktail MGT (4182bp). Together, our study demonstrates the existence of common regulators of different direct reprogramming processes, argues against the premise that TFs possess only lineage-specific capabilities for altering cell fate - the basic premise used to develop direct reprogramming approaches and discover a novel cardiac reprogramming cocktail showing great potential for future clinical application.
Abstract P1009: Size And Distribution Of Human Cardiac Tissue Grafts Following Transendocardial Delivery Of Committed Cardiac Progenitors In A Swine Ischemic Heart Failure Model
Authors: Roy Sushmita , Zhou Tianhua , Schmuck Eric G , Saito Yukihiro , Conklin James , Hacker Timothy A , Hsieh Patrick C , Koonce Chad , Raval Amish N , Kamp Timothy
Published: November 2022

Introduction:
iPSC-derived committed cardiac progenitor cells (CCP) have exciting potential to remuscularize infarcted myocardium; however, little is known about CCP engraftment and differentiation following injection. We hypothesized that transendocardial injection (TEI) of CCPs with an injectable cardiac fibroblast derived extracellular matrix (ECM) retention agent would result in cardiac tissue grafts in an immunosuppressed porcine ischemic heart failure (HF) model.

Methods:
Coronary artery balloon-occlusion myocardial infarction was induced in Yucatan mini-swine. After 1 month, 300M CCPs with and without 50 mg ECM particles were delivered in 15 intramyocardial injections to the border zone and infarct zone using a steerable Myostar injection catheter with NOGA XP mapping. Immunosuppressed swine were sacrificed up to 2 months following treatment. Hearts were systematically sectioned and stained for human nuclear antigen (Ku80) and cardiac troponin I/T to define the area and distribution of human grafts and cardiomyocyte content.

Results:
Clusters of human grafts composed of predominantly troponin positive cardiomyocytes were observed in the infarct, at the infarct border, and in adjacent healthy tissue. Overall, 21/25 (84%) of treated pigs had human cardiac tissue grafts. In the current analysis, grafts were identified in 11/12 (91.6%) and 10/13 (76.9%) of pigs in the CCP+ECM and CCP, respectively (see Figure). The observed mean graft area was 62,930 ± 40,332 μm
2
and 185,200 ± 329,080 μm
2
per pig in the CCP+ECM and CCP only groups, respectively (p=0.23).

Conclusion:
In an immunosuppressed swine ischemic HF model, TEI of CCPs ± ECM yields persistent human cardiac tissue grafts composed of predominantly of cardiomyocytes in both infarcted and non-infarcted regions of the myocardium up to 2 months following treatment.
Abstract P1010: Acetylation Regulates Yap Subcellular Localization In Heart Regeneration
Authors: Liu Shijie , Deshmukh Vaibhav , Meng Fansen , Wang Jun , Martin James
Published: November 2022

The core Hippo pathway effector YAP is a transcription co-factor that directs the expression of genes that promote cardiomyocyte renewal. The Hippo kinases phosphorylate YAP and prevent its nuclear localization, underscoring the importance of post-translational modifications on YAP-mediated transcription. Here, we discovered a lysine acetylation-deacetylation cycle-regulated YAP acetylation in cardiomyocytes. acetylated-YAP is prone to cytoplasmic localization and showed reduced YAP activity compared with WT YAP. In addition, mice that harbor the mutation with abolished YAP acetylation showed better heart regeneration than the controls after myocardial infarction. Thus, our study revealed a novel mechanism that acetylation suppresses YAP activity in cardiomyocytes. Given that there are many clinic-approved compounds targeting acetylation enzymes, we believe our study would guide the development of new strategies targeting YAP acetylation for ischemic heart diseases.
Abstract P1017: Tissue Engineered Human Acellular Blood Vessels For Coronary Artery Bypass Grafting
Authors: Williams Adam R , Nellis Joseph , Wegermann Zachary , McCartney Sharon , Podgoreanu Mihai V , Mehta Sachin , Williams Kyha D , Daubert Melissa A , Kirkton Robert , Nash Kevin , Niklason Laura , Lawson Jeffrey H , Kypson Alan P
Published: November 2022

Objective:
Conduit for coronary artery bypass grafting (CABG) can be limited due to poor quality or prior removal of saphenous veins. A human acellular vessel (HAV) bioengineered from vascular cells and then decellularized for implantation may provide a readily available, off-the-shelf conduit for CABG. Here we evaluate the use of small diameter HAV for CABG in a non-human primate model.

Methods:
Adult male baboons (n=7; 26.8 - 37.0 kg) were imaged by left heart catheterization and computed tomography angiography. CABG was performed using a 3.5 mm diameter HAV to the left anterior descending (LAD) or right coronary artery (RCA) via sternotomy with cardiopulmonary bypass. The proximal coronary artery was ligated to prevent competitive flow and graft transit time flowmetry (TTFM) was assessed after implant. Angiography at 2 weeks, 1, 3, and 6 months evaluated graft patency and heart function. HAVs explanted at 6 months were assessed by immunohistochemistry for host cellular response and remodeling.

Results and Conclusion:
Baboons underwent CABG using a 3.5 mm HAV from the ascending aorta to the LAD (n=2) or RCA (n=5). The diminutive LAD led to early graft loss due to size mismatch and poor runoff. HAV bypass to the dominant RCA (TTFM: 22 ± 7.7 mL/min) resulted in sustained graft patency for up to 6 months post-implantation. HAV explant histology revealed infiltration of multiple host cell populations with formation of a neoadventitial layer and evidence of luminal endothelization. HAV conduits retained structural integrity with no evidence of mechanical failure throughout the study. These results demonstrate that tissue-engineered HAVs may provide an off-the-shelf conduit for CABG.
Abstract P1018: Bone Marrow Progenitor Cell-Derived Exosomes Activate Cardiac Fibroblasts Via Mir-21a-5p Mediated Integrin Alpha V Stabilization
Authors: Ranjan Prabhat , Dutta Roshan Kumar , Zhang Qinkun , Krishnamurthy Prasanna , Lal Hind , Qin Gangjian , Verma Suresh
Published: November 2022

Abstract:
Heart failure is leading cause of death worldwide, and cardiac fibrosis is the hallmark of heart failure. We previously showed that interleukin-10 (IL10) treatment significantly reduces pressure overload-induced cardiac fibrosis. We also have demonstrated that fibroblast progenitor cells (bone marrow-prominin positive cells; FPCs) play a prominent role in developing cardiac fibrosis, yet the mechanism is largely unknown. Here, we hypothesized that pro-fibrotic miRNAs enriched in exosomes derived from IL10 KO FPCs promote fibroblast activation and cardiac fibrosis in pressure-overloaded myocardium.

Methods:
Exosomes were isolated from WT and IL-10KO FPCs. Exosomal miRNAs profiling was performed using fibrosis-associated miRNA profiler kit from Qiagen. To determine the contribution of exosomal miR21 on TAC-induced fibrosis, miR21 level was reduced in FPCs-derived exosomes using miR21 silencer. Modified exosomes were injected into heart immediately after TAC surgery.

Results:
FPCs were identified as CD45+/prominin-1 cells using FACS. TGFβ treatment exacerbated fibrotic genes (Col1α, fibronectin, periostin, and αSMA) expression in IL10KO FPCs compared to WT-FPCs. Pathway-based miRNA array revealed that IL10KO FPCs exosomes are enriched with miR-21a-5p and facilitated fibroblasts activation as observed by qPCR (αSMA and Col1α), western (Col1α and TGFβ levels), wound healing, and immunostaining assay (Col1α/DAPI expression) as compared to WT control. Interestingly, miR21 inhibition in IL10KO FPCs exosome using antimiR 21, reduced TGFβ-induced cardiac fibroblast activation. Target prediction database analysis revealed that Integrin Subunit Alpha V (ITGAV) is a strong regulatory target for miRNA21. At molecular level, we found that exosomal miR-21a-5p stabilizes ITGAV, which ultimately enhances fibroblast activation and cardiac fibrosis both in vitro and in vivo.

Conclusions:
In conclusion, we report that FPCs derived miR21 packaged in exosomes activates cardiac fibroblasts in IL10-KO mice via miR-21a-5p/ITGAV/Col1α signaling axis and increased cardiac fibrosis. IL10 treatment significantly reduced miR21 exosomal packaging, inhibited fibroblasts activation, and reduced cardiac fibrosis following TAC.
Abstract P1027: Cardioprotective Effects Of Genetically Engineered Cardiac Stem Cells By Spheroid Formation On Ischemic Cardiomyocytes
Authors: Jeong Han Saem , Kim Jong-ho , Hong Soon Jun
Published: November 2022

Background:
Sca-1+ cardiac stem cells and their limited proliferative potential were major limiting factors for use in various studies.

Methods:
Therefore, the effects of sphere genetically engineered cardiac stem cells (S-GECS) inserted with telomerase reverse transcriptase (TERT) were investigated to examine cardiomyocyte survival under hypoxic conditions. GECS was obtained from hTERT-immortalized Sca-1+ cardiac stem cell (CSC) lines, and S-GECS were generated using poly-HEMA.

Results:
The optimal conditions for S-GECS was determined to be 1,052 GECS cells/mm
2
and a 48 hours culture period. Compared to adherent-GECS (A-GECS) and S-GECS showed significantly higher mRNA expression of the growth factors SDF-1α and CXCR4. S-GECS conditioned medium (CM) significantly reduced the proportion of early and late apoptotic cardiomyoblasts during CoCl
2
-induced hypoxic injury; however, gene silencing via CXCR4 siRNA deteriorated the protective effects of S-GECS against hypoxic injury. As downstream pathways of SDF-1α/CXCR4, the Erk and Akt signaling pathways were stimulated in the presence of S-GECS CM. S-GECS transplantation into a rat acute myocardial infarction model improved cardiac function and reduced the fibrotic area. These cardioprotective effects were confirmed to be related with the SDF-1α/CXCR4 pathway.

Conclusions:
Our findings suggest that paracrine factors secreted from transplanted cells may protect host cardiomyoblasts in the infarcted myocardium, contributing to beneficial left ventricle (LV) remodeling after acute myocardial infarction (AMI).
Abstract P1035: Co-culturing Human Induced Pluripotent Stem Cell-derived Cardiomyocytes With Mesenchymal Stem Cells Promotes Their Maturation And Enhances Cardioprotection Under Hypoxic Stress
Authors: Sridharan Divya , Ahmed Uzair , Khan Mahmood
Published: November 2022

Introduction:
Co-culturing human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiCMs) with other stem/progenitor cells like bone-marrow mesenchymal stem cells (BM-MSCs) and endothelial progenitors has been shown to improve their survival post transplantation into ischemic hearts. Co-culturing with other cardiac cells has also shown to improve the maturation of hiCMs
in vitro.
However, the potential of co-culturing hiPSC-derived MSCs (hiMSCs) for hiCM maturation and cardioprotection has not been explored.

Hypothesis:
We hypothesize that co-culturing of hiMSCs with hiCMs significantly alters the cells’ proteome, improves hiCM maturation, and enhances cardioprotection.

Results:
hiCMs were co-cultured with hiMSCs for one week at a ratio of 3:1. To evaluate the effect of hiMSCs on hiCM maturation in the co-culture, changes in the hiCM function and gene expression were assessed via multi electrode array (MEA) analysis and qPCR, respectively. MEA analysis showed an increase in ADP50 and ADP90 in addition to improved response to cardiac drugs in hiCMs co-cultured with hiMSCs as compared to hiCM-only cultures, indicating improved maturation of hiCMs in the co-culture. Furthermore, gene expression analysis showed an increased expression of mature CM-associated genes (MYH7, TNNT2). When subjected to hypoxic stress (1% O
2
), we also observed a significant decrease in the number of TUNEL positive cells in the co-cultures as compared to hiCM-only culture. Assessment of hiCM functionality via MEA analysis showed an improved cardioprotection in co-cultures as compared to the single cell-type cultures. Furthermore, proteomic analysis demonstrated increased expression of cardioprotective growth factors (GFs): HGF, VEGFA and IGFBP-1 in the conditioned medium of the co-cultured cells versus single cell-type cultures.

Conclusion:
Overall, our results established that co-culturing hiCMs with hiMSCs improved cardioprotection via increased the expression of cardioprotective growth factors and enhanced the maturation of hiCMs
in vitro.
Abstract P1038: Exosomal RNAs And Proteins From Non-invasively Derived Human Induced Mesenchymal Stem Cells Protect Cardiomyocytes From Death
Authors: Gurusamy Narasimman , Sigamani Vinoth , Perales Selene , RAJASINGH SHEEJA , Rajasingh Johnson
Published: November 2022

Background:
Current stem cells therapy for ischemic cardiomyopathy is effective only about 2-4% in improving cardiac function. In search of novel therapy, we have developed a safe mRNA-based reprogramming of non-invasively derived human urinary epithelial cells into induced pluripotent stem cells (iPSC), and subsequently differentiated them into mesenchymal stem cells (iMSC). We examined therapeutic potential of iMSC in comparison with adult umbilical cord mesenchymal stem cells (MSC).

Hypothesis:
Autologous iMSC and their exosomes possess an enhanced cardioprotective characteristics compared to MSC.

Methods and Results:
The generated iMSC and their exosomes (ExoQuick reagent) offered an enhanced protection of human iPSC-derived cardiomyocytes (iCMC) from injuries of (i) angiotensin-II (Ang, 10 μM for 24 h) and (ii) 6 hrs of 1% hypoxia and 24 hrs of reoxygenation, in comparison with their MSC controls. The cardiomyocyte protection was studied through measurement of mitochondrial membrane potential (JC1 dye), intracellular reactive oxygen species (CM-H2DCFDA), in situ cell death by apoptosis, and qRT-PCR expression of survival and proinflammatory genes (BCL2, BAD, TNFA and IL6). Treatment with iMSC-derived exosomes alone enhanced the expression of NRF2, a major regulator of cytoprotective genes in protecting Ang-challenged iCMC. The specific role of iMSC exosomal RNAs and proteins in mediating cardioprotection against Ang-induced injury was identified through in situ cell death assay using the exosomes treated with either RNase A (0.5 μg/μl for 20 min at 37
0
C) or proteinase K (0.05 μg/μl for 10 min at 37
0
C)
(Fig.1)
. Exosomal noncoding RNA analysis by qRT-PCR revealed the presence of inflammation-associated small nucleolar RNAs such as SNORD32A, SNORD33, SNORD34 and SNORD35A. The transfer of exosomal RNAs into the host cardiac cells was augmented in presence of Ang, identified using 0.5 mM ethynyl uridine-labelled exosomes and detected using Click-iT RNA imaging reagents. All experiments were carried out in triplicates. In vivo studies are in progress.

Conclusion:
In comparison with adult MSC, non-invasively derived autologous iMSC and their exosomes elicit enhanced cardioprotection and may provide an alternative source of cells or cell-free therapy for treating ischemic cardiomyopathy.
Abstract P1041: Redox Regulator MnTnBuOE-PyP5+ Protects Cardiomyocyte Against Oxidative Stress Mediated Myocardial Ischemia/reperfusion Injury
Authors: Sharma Sudha , Bhattarai Susmita , Ara Hosne , Subedi Utsab , Shum Alika , Patel Foram , Batinic-Haberle Ines , Sun Hong , Bhuiyan Md Shenuarin , Panchatcharam Manikandan , Miriyala Sumitra
Published: November 2022

Background:
Much evidence suggests that oxidative stress plays a major role in the pathogenesis of myocardial ischemia/reperfusion (I/R) injury and is a potential target for therapeutic interventions. We tested the hypothesis that the novel redox regulator, MnTnBUOE-2-PyP
5+
(BMX-001), mediates cardioprotection through reduction of oxidative stress in myocardial tissue following I/R injury.

Methods and Results:
BMX-001 (2mg/Kg, IP) or vehicle was administered in mice 4 hours before myocardial ischemia. Mice were then subjected to I/R injury by ligating the left anterior descending artery for 1 hour followed by 24 hours of reperfusion. Following 24 hours of reperfusion, infarct size, cardiomyocyte apoptosis, and SOD2 level were determined, and echocardiography was performed. Our study revealed that the redox regulator provided protection against I/R-induced injury in mice as evident by a significant reduction in infarct size, plasma LDH level, and oxidative stress. In addition, BMX-001 administration in mice markedly increased FoxO3a and SOD2 levels. BMX-001 treatment attenuated I/R induced cardiomyocyte apoptosis in-vivo and in-vitro as evident by a reduction in the number of TUNEL positive cells and improved cell viability. Echocardiography at 24 hours of reperfusion revealed improved ejection fraction and fractional shortening in mice treated with BMX-001. Furthermore, in-vitro treatment of BMX-001 (10μM) 24 hours before hypoxia/reoxygenation in H9c2 cells improved oxidative stress as evident by a reduction in cardiolipin peroxidation, mitochondrial superoxide level, and 4-HNE adducted proteins.

Conclusion:
Pre-treatment with redox regulator BMX-001 showed a beneficial effect in attenuating cardiomyocyte death, mitochondrial superoxide, and cardiolipin peroxidation following I/R injury in H9c2 cells. Pre-treatment with SOD2 mimetic improved heart function, decreased infarct size, and cardiomyocyte apoptosis in mice following I/R injury. Therefore, BMX-001 can be a novel cardioprotective agent against oxidative stress-induced myocardial I/R injury.
Abstract P1054: Oral Administration Of New Agonist Of Adenosine Receptor Reduces Inflammatory Process And Cardiac Remodeling Induced By Myocardial Infarction In Rats
Authors: Nascimento-Carlos Bianca , Da Silva Jaqueline S , Rocha Bruna , Montagnoli Tadeu , Maia Rodolfo , Barreiro Eliezer J , Zapata-Sudo Gisele
Published: November 2022

Treatment of acute myocardial infarction (AMI) is based on strategies to relief pain and increase survival. Thus, this work investigates the effects of a new agonist of adenosine receptor called LASSBio-1860 in an experimental model of AMI, in order to improve cardiac function. The protocols were approved by the Ethics Committee for the Use of Animals at Federal University of Rio de Janeiro (# 103/17). The experimental AMI was induced by the ligation of the anterior descending coronary artery in male Wistar rats (180-200 g), which were randomly divided into groups treated orally with vehicle (DMSO) or 70 μmol/kg of LASSBio-1860 for 7 days. At the end of the experimental protocol, hemodynamic parameters were obtained using echocardiography and the expression of markers involved in the inflammatory pathway (TNF-alpha, p38) and cardiac remodeling (p-ERK-1/2 and t-ERK) was evaluated. AMI increased the left ventricular (LV) wall thickness from 0.11 ± 0.04 to 0.29 ± 0.02 cm (p <0.05) which was reduced to 0.21 ± 0.01 cm after treatment with LASSBio-1860. The impaired ejection fraction observed after AMI (reduction from 91.5 ± 0.5 to 45.2 ± 1.3%) was partially reversed to 60.1 ± 10.3% with the treatment. Additionally, the agonist of adenosine receptor normalized the increased filling pressure of 31.3 ± 4.4 to 15.9 ± 3.9. Increased LV final systolic pressure was detected after AMI, changing from 83.8 ± 12.5 (control group) to 104.7 ± 1.1, which was recovered to 95.3 ± 21.4 mmHg with LASSBio-1860. An increase in the TNF-alpha expression as well as nuclear p38 labeling was detected in the heart from infarcted animals which was reduced after administration of LASSBio-1860. Furthermore, AMI increased the ratio of phosphorylated and total ERK-1/2 from 0.78 ± 0.01 to 0.89 ± 0.01 while treatment with LASSBio-1860 reduced to 0.79 ± 0.01 (p < 0.05). Similarly, p38 increased from 0.72 ± 0.11 to 1.58 ± 0.36 induced by AMI but was reduced to 1.17 ± 0.43 by LASSBio-1860. An increase in collagen deposition from 3.6 ± 0.9 to 31.5 ± 4.29 was observed in hearts from infarcted animals, which was reduced to 26.8 ± 1.6 with treatment. Also, AMI promoted an increase in p38 nuclear labeling from 9.9 ± 3.9 to 40.8 ± 3.3 which was decreased to 29.9 ± 1.7 in treated animals. Similar increase and recovery was detected with iNOS because AMI caused an increase from 9.0 ± 4.1 to 25.3 ± 3.9 and improvement to 14.2 ± 0.6. In conclusion, the reduction of the inflammatory response may be related to the activation of adenosine receptors by LASSBio-1860, which leads to an improvement in AMI-induced cardiac remodeling and dysfunction.
Abstract P1059: Establishing The Mechanistic Basis Of Dilated Cardiomyopathy Associated With The Skeletal Muscle Actin Mutation R256H
Authors: Garg Ankit , Lavine Kory , Jansen Silvia , Greenberg Michael
Published: November 2022

Actin, which is expressed in all eukaryotic cells, is an essential component of the cardiac and skeletal muscle sarcomere thin filament. Mutations in actin have been associated with a wide range of diseases, including skeletal myopathies and cardiomyopathies. Humans express six actin isoforms with two that are thought to be highly enriched in sarcomeres and differ by only four amino acids: skeletal muscle actin (ACTA1) and cardiac actin (ACTC1). While ACTC1 is exclusively expressed in the adult heart, ACTA1 is predominantly expressed in skeletal muscle with lower levels detected in cardiac muscle. As such,
ACTA1
mutations are well known to cause skeletal myopathy with minimal effects on cardiac function. However, I previously reported a family carrying a novel heterozygous mutation in ACTA1 (ACTA1 R256H) with dilated cardiomyopathy (DCM) and no clinical evidence of skeletal myopathy. The objective of this study is to elucidate the molecular mechanism(s) by which ACTA1 R256H may cause heart failure. I generated

ACTA1
R256H/+

human pluripotent stem cells (hPSCs) using Cas9/CRISPR gene editing. Using traction force microscopy, I observed that

ACTA1
R256H/+

hPSC-derived cardiomyocytes display reduced contractility. To define relevant mechanisms, I devised a novel approach to purify milligram quantities of recombinant human ACTA1, which has served as a remarkable challenge for the field. Importantly, this purification technique circumvents contamination from endogenous actins and produces functional protein capable of polymerization. Moreover, in vitro motility assays demonstrated that reconstituted human thin filaments containing recombinant ACTA1 are functional and activated in a calcium-regulated manner. Intriguingly, purified ACTA1 R256H appears to have a polymerization defect, suggesting a possible mechanism by which this mutation leads to a defect in contractility. Together, these studies suggest for the first time that a mutation in skeletal muscle actin, ACTA1 R256H, causes cardiomyocyte hypocontractility while also suggesting a potential molecular mechanism. Finally, this study also introduces a new purification method essential for biochemical and biophysical analysis of human
ACTA1
mutations.
Abstract P1064: Hippo Signaling Pathway Maintains Sinoatrial Node Homeostasis
Authors: Zheng Mingjie
Published: November 2022

The sinoatrial node (SAN) functions as pacemaker of the heart to initiate and drive rhythmic heartbeats. The Hippo signaling pathway is a fundamental pathway for heart development and regeneration. Although abnormalities of Hippo pathway are associated with cardiac arrhythmias in human patients, yet its role in the SAN is unknown. We investigated key regulators of the Hippo pathway in the sinoatrial node by conditionally inactivating the Hippo signaling kinases
Lats1
and
Lats2
using the tamoxifen-inducible, cardiac conduction system (CCS)-specific Cre driver

Hcn4
CreERT2

and
Lats1
and
Lats2
conditional knockout (CKO) alleles. In addition, the Hippo signaling effectors
Yap
and
Taz
were conditionally inactivated in the sinoatrial node. To determine the function of the Hippo signaling pathway in the sinoatrial node and other CCS components, we conducted a series of physiological and molecular experiments including telemetry ECG recording, echocardiography, Masson’s Trichrome staining, calcium imaging, immunostaining, Cleavage Under Targets and Tagmentation (CUT&Tag) sequencing using antibodies against Yap1 or H3K4me3, qRT-PCR, and Western blotting. We found that
Lats1/2
inactivation caused severe sinoatrial node dysfunction (SND; sick sinus syndrome). Compared to the controls,
Lats1/2
CKO mutants exhibited dysregulated calcium handling and increased fibrosis in the sinoatrial node, indicating
Lats1/2
function through both cell-autonomous and non-cell-autonomous mechanisms. Notably, the
Lats1/2
CKO phenotype was rescued by genetic deletion of
Yap
and
Taz
in the CCS, and these rescued mice had normal sinus rhythm and reduced fibrosis of the sinoatrial node, indicating that
Lats1/2
function through
Yap
and
Taz
. CUT&Tag sequencing data showed that Yap regulates genes critical for calcium homeostasis such as
Ryr2
and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as
Tgf-β1
and
Tgf-β3
. Consistently,
Lats1/2
CKO mutants had decreased
Ryr2
expression and increased
Tgf-β1
and
Tgf-β3
expression compared with control mice. We reveal for the first time that the canonical Hippo-Yap pathway has a pivotal role in functional homeostasis of the sinoatrial node. We also provide novel insight into the genetic regulation of sinoatrial node homeostasis and identify new potential therapeutic targets to improve recovery from cardiac arrhythmias.
Abstract P1075: Elucidate The Mechanism Driving The Imbalance Between BMPs And Activin/Gdfs Signaling In Pulmonary Arterial Hypertension
Authors: Chu Kit Yee
Published: November 2022

Transforming Growth Factor-beta (TGF-β) family signaling is involved in Pulmonary Arterial Hypertension (PAH) in humans. PAH is a rare, highly morbid disorder characterized by dysregulated pulmonary vascular remodeling, elevated pulmonary vascular resistance, and the progressive obliteration of the pulmonary arteriolar circulation. The progression of PAH is largely influenced by the proliferation and migration of the pulmonary smooth muscle cells (PASMCs) and pulmonary endothelial cells (PAECs). While the exact mechanism is not understood, there is evidence that germline mutations in the BMPRII gene, one of the TGF-β family type II receptors, is present in PAH patients. Due to the functional similarity of BMPRII and type II Activin receptors ACVR2A and ACVR2B, they interact with overlapping TGF-β family ligands to activate either the SMAD 1/5/8 or SMAD 2/3 signaling. In the pulmonary vasculature, the BMP-9 ligand regulates cell proliferation by binding to type I receptors, ALK-1 and BMPRII, to activate SMAD 1/5/8 signaling. In PAH, there is a decrease in SMAD 1/5/8 signaling and an increase in SMAD 2/3 signaling. However, what causes the signaling switch is still unknown. In our lab, we found out that BMPRII and ACVR2A bind TGF-β ligands with nearly identical geometry based on our crystal structures. Both of the receptors contain a conserved hydrophobic hot spot within the ligand binding pocket. We found that the BMPRII ligand binding domain may exhibit greater flexibility than ACVR2A. We also investigated eight soluble BMPRII extracellular domain mutants and expressed them as BMPRII-Fc fusion proteins (BMPRII-Fc variants). Using surface plasmon resonance, we observed four of the BMPRII-Fc variants bind to high affinity ligands, such as BMP9, BMP10, Activin B and Activin A. In reporter gene assays, we found these BMPRII-Fc variants act as a ligand trap and attenuate BMP9 and BMP10-mediated SMAD1/5/8 signaling in HEK293 cells. Interestingly, they did not inhibit Activin B-mediated SMAD2/3 signaling. In conclusion, our findings show gain of function of specific BMPRII mutations and suggest that imbalances between SMAD1/5/8 and SMAD2/3 signaling may lead to PAH progression.
Abstract P1094: Sexual Dimorphism In Histone Deacetylases Activity Regulates Ventricular Calcium Handling Properties
Authors: Almeida Jonathas F , Banach Kathrin
Published: November 2022

Introduction:
There is a sexual dimorphism in cardiac excitation contraction coupling (ECC) with female ventricular myocytes (F
VM
) exhibiting an attenuated basal calcium concentration ([Ca]
i
), Ca transient (CaT) amplitude, and a prolonged CaT decay constant (τ). Increased expression levels of the sodium-calcium exchanger (NCX) have been described in F
VM
however, the role NCX expression plays in sexual dimorphism of physiological and pathophysiological Ca handling properties, and how NCX expression is regulated in a sex specific manner remains to be determined. Here we tested the hypothesis, that a sexual dimorphism in HDAC activity regulates NCX expression and NCXs differential contribution to the CaT adds to the sexual dimorphism in ventricular ECC.

Method:
[Ca]
i
was quantified in field stimulated, freshly isolated ventricular myocytes (VMs) from male (M
VM
) and female (F
VM
) FVB/N mice loaded with the Ca sensitive fluorescent dye Fluo-4/AM. Changes in NCX expression were quantified by qPCR and western blotting.

Results:
Under basal conditions F
VM
exhibited an attenuated CaT amplitude (ΔF/F
0
; M
VM
:1.8±0.08, F
VM
:1.4±0.06, p=0.038) and prolonged CaT decay constant (τ: M
VM
:0.37±0.01ms, F
VM
:0.46±0.02ms, p<0.01). The caffeine induced Ca transient (CaT
caf
) amplitude was comparable in F
VM
and M
VM
, but the CaT
caf
decay constant (τ
caf:
M
VM
: 3.34±0.22s
;
F
VM
:4.81±0.36s) was longer in F
VM
although increased NCX expression was determined on the protein and mRNA level. Consistent with increased NCX expression, F
VM
showed an attenuated increase in basal [Ca]
i
(F/F
0
; M= 60%, F=44%, p=0.0033) and τ (M=0.37±0.01s, F=0.46±0.02s, p<0.0001) when SERCA activity was attenuated (CPA: 10μM). Also during conditions favoring NCX reverse mode ([Na]
o
=93, 46 mM) F
VM
exhibited a larger increase in CaT amplitude (ΔF/F
0
; M=81%, F=133%, p<0.01) and an increased propensity for spontaneous Ca release events. Inhibition of HDAC class II (LMK: 5mg/kg/day) did not change Ca handling properties in F
VM
but slowed τ (M
LMK
: 0.44±0.03ms) and τ
caf
in M
VM
(M
LMK
=5.1±0.33s) making them comparable to F
VM
.

Conclusion:
The sexual dimorphism in ventricular ECC is mediated by attenuated HDAC activity in F
VM
s and facilitates NCX Ca overload.
Abstract P1104: Lower Troponin Expression In The Right Ventricle Of Rats Explains Interventricular Differences In E-c Coupling
Authors: Jeon Young Keul , Kwon Jae Won , Kim Sung Joon
Published: November 2022

Despite distinctive functional and anatomical differences, a precise understanding of the cardiac interventricular differences in excitation-contraction (E-C) coupling mechanisms is still lacking. Here, we directly compared rat right and left cardiomyocytes (RVCM and LVCM). Whole-cell patch clamp, IonOptix system and fura-2 fluorimetry were used to measure electrical properties (action potential and ionic currents), single cell contractility and cytosolic Ca
2+
([Ca
2+
]
i
), respectively. Myofilament proteins were analyzed by immunoblotting. RVCMs showed significantly shorter action potential duration (APD) and higher density of transient outward K
+
current (
I
to
). However, the triggered [Ca
2+
]
i
change (Ca
2+
transient) was not different, while the decay rate of the Ca
2+
transient was slower in RVCM. Although the relaxation speed was also slower, the sarcomere shortening amplitude (ΔSL) was smaller in RVCM. SERCA activity was approximately 60% lower in RVCM, partly responsible for the slower decay of the Ca
2+
transient. Immunoblot analysis revealed lower expression of the cardiac troponin complex (cTn) in RVCM, implying a smaller Ca
2+
buffering capacity (
κ
S
), which was proved by
in situ
analysis. The introduction of these new levels of cTn,
I
to
and SERCA into a mathematical model of rat LVCM reproduced the similar Ca
2+
transient, slower Ca
2+
decay, shorter APD and smaller ΔSL of RVCM. Taken together, we show reduced expression of cTn proteins in the RVCMs, which provides an explanation for the inter-ventricular difference in the E-C coupling kinetics.
Abstract P1109: Investigating The Role Of Human Conserved Microrna-193 In The Aging
Drosophila
Heart
Authors: Hohman Alyssa M , McNeill Elizabeth M
Published: November 2022

MicroRNAs (miRNAs) are short, non-coding, RNAs that post-transcriptionally regulate gene expression and are ideal candidates for the regulation of the genes responsible in the heart. Profiling studies have shown circulating miRNAs to be implicated in human cardiovascular disease with the potential to be used as biomarkers for various cardiovascular pathologies. Highly conserved with
Drosophila
miR-193, human miR-193 is expressed in the heart and has been established as a biomarker to distinguish between patients with different types of heart failure, including HFPEF, hypertrophic cardiomyopathy, ischemia reperfusion injury, right ventricular remodeling, cardiac fibrosis, and congenital heart disease. However, without the mechanism and regulation of its target genes, the use of miR-193 as a biomarker or therapeutic is limited. We have characterized the impact of miR-193 loss on homoeostasis of heart morphology and function in
drosophila
during aging using a combination of null mutants and heart specific dysregulation of miR-193 expression. Using SOHA (semi-automated optical heartbeat analysis), we observed changes occurring to heart structure and function with a loss of miR-193. With miR-193 dysregulation, significant changes in heart rate, heart size (diastolic and systolic diameters), arrhythmia index, and diastolic/relaxation interval time, began at 10 days post-eclosion and continued as the flies aged. Understanding the role of miRNAs in specific tissues and disease states will ultimately depend on our ability to dissect the fundamental strategies and mechanisms by which miRNAs regulate cardiac biology. Using
in silico
analysis, we have identified multiple human conserved putative miRNA-target gene interactions to further characterize the molecular mechanisms underlying the observed phenotypes. Our results suggest that the human conserved
Drosophila
miR-193 is essential for normal cardiac function. In miR-193 loss of function, we see significant changes to heart rate, heart size, and interval time consistent with an early aging phenotype. The conservation of miR-193 and its predicted gene targets enable us to discover the molecular mechanisms that underlie miRNA dysregulation in cardiovascular disease.
Abstract P1112: Inhibition Of Wnt Signaling Protects Against Myxomatous Valve Disease In A Mouse Model Of Marfan Syndrome
Authors: Xu Na , Yutzey Katherine E
Published: November 2022

Introduction:
Myxomatous valve disease (MVD) is the most common cause of mitral regurgitation, characterized by valve leaflet thickening and progressive valve degeneration, leading to impaired cardiac function and heart failure. MVD in a mouse model of Marfan syndrome (MFS) includes valve leaflet thickening and increased immune cell infiltration. However, the underlying mechanisms by which immune cells are recruited in MFS valves remain undefined.

Hypothesis:
Inhibition of Wnt signaling prevents immune cell recruitment and MVD progression in MFS valves.

Methods:
Mice with the mutation of Fibrillin 1 (

Fbn1
C1039G/+

) recapitulate histopathological features of MFS and Wnt signaling activity was detected in TCF/Lef-lacZ reporter mice. Inhibition of Wnt signaling was achieved by conditional induction of the soluble Wnt inhibitor Dkk1 in periostin-expressing valve interstitial cells of Periostin-Cre; tetO-Dkk1; R26rtTA; TCF/Lef-lacZ;

Fbn1
C1039G/+

mice. Dietary doxycycline was administered for one month beginning with MVD initiation (1-month-old) or MVD progression (2-month-old). Histological evaluation and immunofluorescence for immune cells were performed.

Results:
Wnt signaling as detected by TCF/Lef-reporters is activated early in mitral valve disease progression, prior to immune cell infiltration in

Fbn1
C1039G/+

mice. Inhibition of Wnt signaling by Dkk1 induction at 1-month of age reduced valve leaflet thickness with decreased CD45+ leukocytes in

Fbn1
C1039G/+

mice. However, later Wnt inhibition starting at 2 months did not alter valve leaflet thickness.

Conclusions:
Inhibition of early Wnt signaling ameliorates myxomatous valve degeneration and immune cell infiltration previously demonstrated to be required for progression of MVD.
Abstract P1123: Establishment Of An In Vitro Method To Evaluate Chronic Cardiac Contractility Modulation Signals In 3d Human Engineered Cardiac Tissues
Authors: Feaster TK , Ewoldt Jourdan , Casciola Maura , Narkar Akshay , Chen Christopher S , Blinova Ksenia
Published: November 2022

Background:
Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical simulations to the heart during the absolute refractory period to enhance cardiac function. We previously evaluated the acute effects of CCM in isolated rabbit-CM and 2D hiPSC-CM monolayers, on flexible substrate, and found enhanced calcium and contractility. In the present study, we sought to extend these results to chronic studies in 3D human engineered cardiac tissues (ECTs) to develop a robust model to evaluate the long-term effects of CCM stimulation
in vitro
on intact human cardiac tissues.

Methods:
HiPSC-CMs and cardiac fibroblasts in a fibrin-based gel were combined to form ECTs (Figure 1). Morphology and contractility were evaluated. We found ECTs displayed baseline peak contraction amplitude of 194.8±80.8 a.u., (n=5) and peak force and stress of 26.5±6.8 uN, (n=5) and 87.9±22.0.8 Pa, (n=5) respectively. ECT displayed robust baseline electrophysiological (AP) properties and calcium handling properties. Chronic CCM pulses (i.e., 30 minutes) were applied using a commercial pulse generator. Under these conditions ECTs displayed enhanced contractile kinetics 29.0±0.04 %, (P<0.01, n=4), relaxation kinetics 23.0±0.05 %, (P<0.05, n=4), and shorter duration 32.0±0.03 %, (P<0.01, n=4) relative to time matched control.

Conclusion:
This study provides a comprehensive characterization of chronic CCM effects on 3D ECTs. Future studies will investigate prolonged time points. These data provide an
in vitro
model to assess physiologically relevant mechanisms and evaluate safety and efficacy of future cardiac electrophysiology medical devices.

Disclaimer:
The mention of commercial products, their sources, or their use in connection with material reported herein is not to be construed as either an actual or implied endorsement of such products by the Department of Health and Human Services.