bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1108: Development Of A Vascularized Cardiac Patch Using 3D Bioprinting To Regenerate Injured Myocardium
Authors: Hwang Boeun , Korsnick Lauren , Shen Ming , Bauser Heaton Holly , Serpooshan Vahid
Published: October 2023

Introduction:
Regeneration of the myocardium after ischemic injuries has remained a challenge due to the low proliferative capacity of cardiomyocytes (CMs). With the advance of tissue engineering, various cardiac patch devices have been developed and shown to improve cardiac tissue structure/function after myocardial infarction (MI). However, the therapeutic outcomes of patch devices have been limited by the lack of biological and structural complexity. Leveraging 3D bioprinting and stem cell technologies, this study presents the development of a next generation cardiac patch, integrated with heterogenous multi-cellular architecture, perfusable vasculature, and pro-proliferative molecules.

Methods:
A hybrid functional bioink, consisting of gelatin methacrylate (GelMA), gelatin, and fibrin, was optimized to fabricate the patch constructs. Two small molecule-based CM expansion techniques were employed, utilizing follistatin-like protein 1 (FSTL1) and CHIR99021 (CHIR), to further enhance the regenerative capacity of the patch. Complex vascular geometries were created by bioprinting sacrificial pluronic bioink in between two cellular composite bioink layers, containing human induced pluripotent stem cell-derived CMs (hiPSC-CMs) and endothelial cells (ECs). The sacrificial ink was subsequently removed to create vascular channels, which were then endothelialized with ECs.

Results:
Bioprinted patches were successfully cultured under static and dynamic (physiologic flow) culture
in vitro
. The hiPSC-CMs encapsulated in gelMA-gelatin-fibrin bioink showed adequate long-term viability and function within the 3D environment. The printed vascular channels formed rather uniform endothelium. Both the FSTL1 and CHIR treatments resulted in significant in-situ expansion of hiPSC-CMs within bioprinted cardiac constructs. Further, testing the engineered patch in a rat MI model demonstrated the significant cardioprotective effect of patch.

Conclusion:
Together, this study introduces a new generation of bioprinted myocardial tissue analogues as a robust research-enabling platform for
in vitro
testing, and as a novel patch device with markedly enhanced regenerative capacity to treat diseased/damaged adult mammalian heart.
Abstract P1113: Patient-derived Cardiac Assembloids Enable Therapeutic Verification Of Regenerative Reagent Bio
Authors: Kim Yong-Sook , Cho Meeyoung , Cho Dong Im , Yoo Sooji , Ahn Youngkeun
Published: October 2023

Background:
Preclinical study is a prerequisite for drug developments, and yet animal models are poor predictors in humans. We previously reported GSK3β inhibitor BIO (6-Bromoindirubin-3’-oxime) induced cardiac regeneration in zebrafish and rat models.

Methods and Results:
For further development of BIO in the models with human features, we generated human pluripotent stem cells (h-iPSCs) to get the cells which are components of heart. H-iPSCs were derived from blood mononuclear cells, or generated from fibroblasts isolated from the heart tissues of recipients after transplantation or from heart samples during autopsy. After confirming the cell markers, h-iPSCs were differentiated into cardiomyocytes (CM), cardiac myofibroblasts (MF), and macrophages (Mac). Decellularized extracellular matrix (dcECM) were extracted from hearts, and we developed spheroids with various combinations. Since CM hardly proliferate and excessive MF is accumulated in damaged heart, cardiac regeneration is challenged by reversal them. Firstly, the effective dosage of BIO was 0.5uM on human cells, while was 5uM on cardiac cells of mice and rats. On BIO treatment with 0.5μM for 3 days, the numbers of total CM and EdU-or Ki67-positive CM were higher in the BIO-treated CM spheroids. Conversely, viability, migration and collagen gel contraction activities of MF were substantially inhibited by BIO treatment.

Conclusions:
Because tremendous trials for regeneration have failed in clinical applications, it should be validated in realistic study designs with human cells. Here, we successfully reproduced the effects of BIO on human cardiac cells, and suggest the reciprocal regulation of CM and MF by BIO could be attractive therapeutic to heart diseases. More targeted approaches using human cardiac cells and dcECM might allow specific targeting of cardiovascular disease thus optimize regenerative therapy.
Abstract P1114: Preclinical Large Animal Assessment Of Safety And Engraftment Of Hicms Nanofiber Cardiac Patch Transplantation For Cardiac Repair
Authors: Sridharan Divya , Baseeruddin Syed , Mergaye Muhamad , Simonetti Orlando , Varghese Juliet , Han Yuchi , Hamlin Robert , Powell Heather , Khan Mahmood
Published: October 2023

Background:
Myocardial infarction (MI) is a leading cause of death in the United States. However, none of the therapies can regenerate the damaged heart muscle.

Objective:
The objective of this study was to assess the safety and engraftment of a nanofiber cardiac patch laden with human iPSC-derived cardiomyocytes (hiCMs) and transplanted in a preclinical porcine MI model.

Methodology:
MI was induced in pigs via advancing a balloon catheter from the femoral artery into the left anterior descending coronary artery (LAD) below the first diagonal branch (D1) and the balloon was inflated for 90-min to induce myocardial ischemia. Immunosuppressant drug Tacrolimus was given orally one-week before the cardiac patch transplantation and continued for up to 4-weeks post-cardiac patch transplantation. Epicardial transplantation of aligned nanofiber cardiac patch (2 layers) loaded with hiCMs was performed at one-week post MI. Magnetic resonance Imaging (MRI) was performed prior to the MI induction (Baseline), 1-week post-MI and at 4-weeks post cardiac patch transplantation to assess severity of MI and cardiac function. At 4-weeks post-MI, pigs were euthanized, and the heart tissues were processed for immunostaining to confirm the retention and engraftment (Human Nuclear Antibody staining) of the implanted cells in the host myocardium.

Results: (a)
A significant increase in ST-elevation following 90-min of MI,
(b)
Incidence of cardiac arrhythmias were not observed post cardiac patch transplantation, confirming the safety of cardiac patch,
(c)
No teratomas were observed at the transplanted site, indicating safety of terminally differentiated hiCMs and
(d)
Immunofluorescence staining of cardiac sections in the transplanted cardiac patch area confirmed engraftment of transplanted cells via anti-human nuclear antibody, which demonstrates the survival and engraftment of implanted cells.

Conclusions:
In summary, we have demonstrated for the first time that aligned nanofiber cardiac patch loaded with hiCMs is safe and can enhance retention of transplanted cells. Further studies are warranted to assess the long-term efficacy of cardiac patch transplantation in improving the cardiac function and to perform first-in-human clinical trials in the near future.
Abstract P1121: Substrate Rigidity And Mybpc3 Genotype Regulate Development Of Hypertrophic Cardiomyopathy Structural And Functional Phenotypes In Human Ipsc-derived Micro-heart Muscle Arrays
Authors: Guo Jingxuan , Jiang Huanzhu , Schuftan David , Aryan Lavanya , Huebsch Nathaniel
Published: October 2023

Introduction:
We hypothesized that mechanical loading is a key factor in the early stages of hypertrophic remodeling associated with pathogenesis of hypertrophic cardiomyopathy (HCM). We tested this hypothesis
in vitro
with human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) bearing heterozygous loss-of-function mutations in Myosin Binding Protein C (MYBPC3). Cardiomyocytes were cultured in engineered micro-heart muscle arrays that were loaded by changing substrate rigidity.

Methods:
Isogenic control and MYBPC3
+/-
hiPSC-CM were seeded into dogbone-shaped molds to form micro-heart muscle atop silicone rubber substrates with rigidity levels ranging from levels of embryonic (0.4 kPa) to fibrotic adult heart (114 kPa). Spontaneously contracting micro-tissues formed within 48 hours. 10 days after formation, tissues were monitored for action potentials (voltage sensitive dye, BeRST-1), Ca
2+
transients (GCaMP6f reporter), and contractility (substrate deflection). Cell and tissue architecture were assessed in 8 μm cryosections, and RNA transcription via qRT-PCR.

Results:
MYBPC3
+/-
and control cardiomyocytes had similar physiology and morphology in 2D monolayers. However, MYBPC3
+/-
tissues exhibited marked changes in Ca
2+
handling, which were exacerbated when substrate stiffness increased to the level of healthy heart muscle (15 kPa). This was concurrent with hypertrophy of MYBPC3
+/-
cardiomyocytes at 15 kPa. MYBPC3
+/-
tissue Ca
2+

handling abnormalities were linked to enhanced Ca
2+

intake rather than SERCA deficiency or excessive myofilament buffering. MYBPC3
+/-
tissues exhibited mis-regulation of cardiac troponins on the RNA and protein localization levels, which may underlie the observed structural and physiological changes.

Conclusions:
Changes in the rigidity of the substrate, mimicking changes in heart muscle caused by blood pressure, pushed hiPSC-cardiac micro-tissues with MYBPC3 mutations to recapitulate key structural and electrophysiological characteristics of HCM. In contrast, control tissues exhibited hypertrophy and abnormal calcium handling, but only at extreme values of substrate stiffness. This suggests that HCM mutations may exaggerate load-dependent hypertrophic remodeling.
Abstract P1122: Ablation Of PDGFRβ Expression In Cardiac-resident Mesenchymal Stem Cells Prevents Adverse Cardiac Remodeling In Chronic Heart Failure
Authors: Hamid Tariq , Ismahil Mohamed Ameen , Liu Wei , Zhu Yujie , Katsnelson Michael , Ma Lina , Mark Mengya , Rokosh Gregg , Prabhu Sumanth D
Published: October 2023

Failing hearts exhibit chronic inflammation and progressive fibrosis. We recently reported temporal expansion and a pro-fibrotic role of Sca1
+
cardiac mesenchymal stem cells (cMSCs) in chronic heart failure (HF), that was in part related to their increased expression of platelet derived growth factor (PDGF) receptor beta (PDGFRβ). PDGF signaling is mediated by two distinct receptors; -alpha (PDGFRα) and PDGFRβ. Whereas PDGFRα is considered a MSCs marker, the specific role of cMSC-localized PDGFRβ in failing hearts is unknown. We
hypothesized
that augmented cMSC-PDGFRβ expression in chronic HF promotes maladaptive LV-remodeling, fibrosis, and inflammation. We generated Sca1
+/MerCreMer
-PDGFRβ
f/f
transgenic mice (Tg) with Sca1-directed inducible ablation of PDGFRβ expression. Littermates without Cre expression were used as controls. In our preliminary studies, following 2 w of permanent coronary ligation, mice (7-10/group) were fed tamoxifen-containing chow for 13 w. Echocardiography revealed that at 13 w, control mice had significant LV dilatation (ESV 117 ± 59 vs. 64 ± 37.6 μL; EDV 154 ± 69 vs. 87 ± 41 μL) whereas, LV dilatation in Tg mice was stabilized (ESV 78 ± 38 vs. 69 ± 18 μL, EDV 112 ± 42 vs. 92 ± 22 μL), with enhanced cardiac function (EF 32 ± 12 vs. 22 ± 7%). Analysis of changes in ESV, EDV, and EF revealed significantly smaller ΔESV, ΔEDV, and increased ΔEF, in Tg mice. Gravimetric data indicated Tg mice with significantly decreased normalized heart, spleen, and wet lung weights, all indicative of attenuated HF progression. Flow cytometric assessment of inflammatory responses indicated a significant decrease in; 1) circulating Ly6c
hi
monocytes, 2) total myocardial MHCII
+
F4/80
+
macrophages, and, 3) F4/80
+
CCR2+ infiltrating macrophages in Tg mice. These effects in Tg mice occurred together with a significant decrease in remote zone fibrosis (2 ± 2 vs. 9 ± 8%) and myocyte hypertrophy (422 ± 62 vs 527 ± 34 μm
2
). We
conclude
that ablation of cMSC-PDGFRβ expression in post-infarction HF has beneficial effects that may be attributed to attenuation of LV-remodeling and dysfunction and a subsequent contraction of both systemic and myocardial inflammation. Modulation of PDGFRβ expression could thus be exploited as a therapeutic intervention in chronic HF.
Abstract P1140: Acute Mechanosensitive Adaptation To Phenylephrine In Cardiomyocytes And Fibroblasts
Authors: Kimball Todd H , Gromova Tatiana , Chapski Douglas , Vondriska Thomas M
Published: October 2023

Mechanosensitive pathways are fine tuned to the physiological environment of the cell. Adrenergic stimulation induces cell type specific responses in the heart, including cardiomyocyte hypertrophy and fibroblast proliferation. We hypothesized acute induction of hypertrophy by phenylephrine (PHE) would activate mechanosensitive pathways in fibroblasts and myocytes, revealing novel molecular features of early hypertrophic adaptation. Male mice were subcutaneously injected with 20 mg/kg of PHE or PBS every other day for one week. Echocardiography revealed significant cardiac hypertrophy with increased ejection fraction (72.45%
+
6.15 PHE v 56.32
+
5.32 PBS, p<0.0001) and LV wall thickness (2.25 mm
+
0.29 PHE v 1.62
+
0.13 PBS, p<0.0001), along with diastolic impairment, as measured by elevated E/e’ ratio (37.25
+
7.29 PHE v 23.67
+
3.62 PBS, p<0.0001). Interestingly, histological analysis showed increased fibrotic deposition and cardiomyocyte cross-sectional area was preferential increased in fibrotic areas (265.5 um
2
+
67.5 PHE fibrotic v 195.1
+
47.46 PHE non-fibrotic v 165.6
+
46.96 PBS). While cardiac fibroblasts from PHE-treated animals did not show alpha smooth muscle actin staining, a traditional marker for activated myofibroblasts, fibroblasts from PHE-treated mice showed increased f-actin stress fibers and nuclear localization of Mrtfa, a mechanosensitive transcriptional coactivator. We performed RNA-seq on isolated cardiomyocytes, demonstrating activation of myocyte-specific responses to increased pressure (
Des, Nppa
), as well as activation of fibrotic genes (
Tgfbr1, Tgfbr2, Tgfb2
)
in myocytes
. We sought to investigate mechanosensitive pathways initiated by PHE treatment in cardiomyocytes and identified transcriptional changes in Ankrd1, a Titin binding protein which translocates to the nucleus upon stress. We demonstrate a PHE induced increase in Ankrd1 protein expression and nuclear localization (nuclear area occupancy, as measured by microscopy, 17.04%
+
8.76 PHE v 11.91%
+
9.134 PBS, p=0.002) in cardiomyocytes. Together these findings demonstrate distinct acute phenotyptic adaptation to adrenergic stress in myocytes and fibroblasts, revealing cell type-specific mechanosensitive signaling mechanisms.
Abstract P1141: Molecular Insights Into The Phenotypic Heterogeneity Observed In Two Different Murine Models Of Hypertrophic Cardiomyopathy (HCM)
Authors: Thottakara Tilo , Long Carlin S , Padmanabhan Arun , Olgin Jeffrey , Abraham M Roselle
Published: October 2023

Background:
Phenotypic variability in HCM has long been appreciated to correlate with genotype. However, the molecular drivers underpinning these differences remain poorly understood. We hypothesized that the variable HCM phenotypes observed in 2 different widely used HCM mouse models (R403Q-MyHC, R92W-TnT) result from complex interactions between a variety of cardiac cell types. We performed unbiased assessments of gene expression and chromatin accessibility in mutant and littermate control hearts at an early disease stage, when MyHC-mutants have higher LV-mass and normal LV-function, while TnT-mutants have similar LV-mass, hypercontractility and diastolic dysfunction, compared to littermate-controls.

Methods:
Nuclei were isolated from hearts of 5-week-old mutant and control animals and subjected to the standard linked ATAC and gene expression profiling pipeline from 10X Genomics w/o enrichment/sorting. Clustering, differential gene expression and transcription factor (TF) motif enrichment was performed using Seurat, ArchR; SCENIC was used to predict TF (Regulon)-activation from gene expression data and Cellchat for cellular crosstalk.

Results:
Integrated single-nuclei data-based clustering identified all cardiac cell types. Both mutant ventricular(V)-myocytes had similar dysregulation (80-90% agreement) of gene expression and TF enrichment, with matching of 23/30 top TF-motifs, and agreement with gene expression-based TF-prediction. Both mutant V-myocytes had activation of SWI/SNF complex TFs (Jun, Fos, Smarc family), however activation of SREBF-1/2, Atf5, Creb3l2, Egr2 (drivers of fibroblast activation), and Klf2, Klf4, Bcl6b (drivers of shear stress-mediated gene expression in endothelial cells (ECs)) was only present in TnT-mutants. Activation of VCAM signaling from V-myocytes-to-ECs, and PDGF signaling to VSMCs is predicted in both mutants, but TGF-β signaling to fibroblasts, endothelin-1 signaling to V-myocytes, was only predicted in TnT-mutants.

Conclusions:
Mutant sarcomeric proteins activate the SWI/SNF complex that drives V-myocyte hypertrophy at early disease stage. Mutation-specific activation of TF-programs in non-myocyte cells are important drivers of cardiac phenotypic variability in HCM.
Abstract P1146: Global Analysis Of Murine Cardiac Gene Program Regulated By Thyroid Hormone And Transcriptional Repressor Rest
Authors: Russell Megan L , Wu Bingruo , Lu Pengfei , Zheng Deyou , Zhou Bin
Published: October 2023

Background:
During adult heart failure, transcriptional reprogramming of fetal cardiac genes coincides with dysregulated heart functions. However, the underlying mechanisms remain incompletely understood. Thyroid hormone and RE1 silencing transcription factor (REST) are known regulators of cardiac gene reprogramming. Here, we report a global analysis of their co-regulatory functions.

Methods:
We generated a dual
Myh6
and
Myh7
mouse reporter line. This line was crossed with a conditional
Rest
allele to generate myocardial
Rest
knockout mice. Control and
Rest
null mice were treated with propylthiouracil (PTU) to inhibit thyroid hormone synthesis. PTU treatment induced heart failure and adult-to-fetal cardiac gene remodeling. Single nuclei multiomic profiling was performed, linking gene expression and gene regulatory network information from each nuclei to elucidate the impact of PTU or REST or both on cardiac gene reprogramming. Samples were collected to validate the newly identified factors involved in reprogramming.

Results:
Data analysis showed that PTU treatment upregulates transcription factor binding involving circadian gene expression (
Cry2
,
Per2
,
Per3
). Downregulated genes are related to voltage-gated channel activity and BMP/TGFB activities involving heart development (
Myh6
,
Nrg1
,
Bmpr1a
) and SA node action potential (
Scn5a
,
Hcn2
,
Ryr2
). In contrast,
Rest
knockout upregulated genes are associated with arrhythmia,
i.e.
, cAMP catabolism (
Pde4d
,
Pde7a
,
Pde7b
), heart conduction (
Cacna1c
,
Gja1
,
Pln
), and ion transport (
Kcnip2
,
Pde4d, Esr1
). Downregulated genes are involved in protein functions involving integrin-mediated signaling (
Itgal
,
Itga4
,
Itga5
), cytoskeleton organization (
Ctnna2
,
Actb
,
Xirp1
), and fiber organization (
Tgfb3
,
Smad3
,
Ptk2b
). Furthermore, a subset of genes was found to be co-regulated by thyroid hormone and REST, including
Ccn2
,
Myh9
,
Nmrk2
,
Fgr
,
Itga4
,
Prkcb
,
Cspg4
which are involved in integrin signaling pathway, immune activation, and angiogenesis.

Conclusion:
Thyroid hormone and REST have shared function in cardiac gene reprogramming involving cardiac conduction and fibrosis as well as their own newly discovered, unique roles regulating circadian genes and cAMP catabolism, respectively.
Abstract P1153: ANT-Dependent MPTP Contributes To Necrotic Myofiber Death In Muscular Dystrophy
Authors: Bround Michael J , Havens Julian , York Allen , Sargent Michelle , Molkentin Jeff D
Published: October 2023

The Mitochondrial Permeability Transition Pore (MPTP) is a critical cell death mechanism that contributes to ischemia reperfusion injury in the heart and several degenerative diseases, including Muscular Dystrophy (MD). MD is a family of genetic disorders characterized by progressive muscle wasting, loss of muscle function, and premature death. We have recently demonstrated that the MPTP has two defined genetic components: one consisting of adenosine nucleotide translocase (ANT) family of proteins and a second, unidentified pore that requires the protein cyclophilin D (CypD) to activate. In this study we used MD as a model to test whether this new MPTP model could be applied to a physiological context of necrotic cell death
in vivo
. We observed that genetic deletion of ANT1 or CypD substantially reduced MD pathology in skeletal muscle, with reduced measures of muscle necrotic cell death, and improved muscle function compared to MD control animals. However, both ANT1KO and CypDKO MD mice still displayed elevated rates of cell death and MD pathology compared to healthy wildtype animals. MD mice lacking both ANT1 and CypD (ANT1KO-CypDKO) virtually eliminated features of skeletal muscle pathology in MD. We observed a near complete loss of MD histopathology, undetectable levels of tissue necrosis, and muscle function that was improved to non-MD levels in ANT1KO-CypDKO MD mice. This study demonstrates that inhibiting both the ANT-dependent and CypD-dependent components of MPTP together can prevent necrotic cell death in an
in vivo
model. This study also suggests that a therapy that inhibits both MPTP-components can effectively treat MD and may potentially reduce cardiac ischemia reperfusion injury.
Abstract P1154: Loss Of Bcl-Rambo Is Protective Against Ischemia-Reperfusion Injury
Authors: Mendoza Arielys , Karch Jason , Patel Pooja J , Robichaux Dexter
Published: October 2023

Myocardial infarction (MI) is a leading cause of mortality worldwide. Various forms of cell death have been implicated to occur during MI, including apoptosis, necrosis, and autophagic cell death. Unfortunately, death of cardiomyocytes (CMs) in the adult heart is permanent, which demonstrates a need to therapeutically regulate death pathways during MI. To identify novel cell death regulators, we performed a screen in which we exogenously expressed over 13,000 human cDNAs into murine fibroblasts, which were subsequently challenged by a necrotic insult. From these screening efforts, we identified Bcl-Rambo, a B-cell lymphoma 2 (Bcl-2) family member, as a protective regulator against necrosis. We also determined that Bcl-Rambo is the most abundantly expressed Bcl-2 family member in wild-type (WT) mouse heart mitochondria. However, whether Bcl-Rambo is pro- or anti-apoptotic remains controversial in the literature. Recent studies have indicated Bcl-Rambo is a mediator of mitophagy, yet it is unclear how this is connected to its involvement with cell death. To determine the role of Bcl-Rambo in the heart, we generated a novel Bcl-Rambo genetic deletion mouse model and determined that key regulators of mitophagy and mitochondrial fusion are reduced. Additionally, we found that total levels of autophagy are severely blunted in the Bcl-Rambo null heart compared to WT hearts. Furthermore, Bcl-Rambo null mouse embryonic fibroblasts (MEFs) exhibit elongated mitochondria and are susceptible to multiple forms of cell death. Surprisingly, after subjecting these mice to I/R injury, we observed that Bcl-Rambo null hearts exhibit significantly smaller infarct size than their WT counterparts, suggesting that Bcl-Rambo expression is deleterious in this model. In summary, Bcl-Rambo is a unique Bcl-2 family member that regulates mitochondrial dynamics, mitophagy, and cell death. Identifying the diverse roles that Bcl-Rambo plays in mitochondrial health is critical for clinically targeting pathways during MI.
Abstract P1162: Nedd4-2 Inactivation Underlies The Increase In Cardiac Sodium-dependent Glucose Cotransporter-1 In Diabetic Hearts
Authors: Pandey Vivek K , Velmurugan Sathya , Despa Sanda
Published: October 2023

Rationale:
Type-2 diabetes (T2D) is a major risk factor for developing heart failure. While the underlying mechanisms are poorly understood, recent evidence suggests that upregulation of the Na
+
/glucose cotransporter 1 (SGLT1) plays an important role, as it causes myocyte Na
+
overload.

Objective:
To identify the mechanisms involved in promoting an increase in SGLT1 protein level in diabetic hearts.

Methods/Results:
We compared hearts from patients with and without T2D and hearts from rats with late-onset T2D caused by overexpression of human amylin in pancreatic β-cells versus hearts from wild-type littermates. SGLT1 protein level was significantly higher in hearts from both humans and rats with T2D. In contrast, the SGLT1 mRNA levels were comparable in hearts from T2D and non-diabetic patients and rats. This result suggests that theT2D-induced increase in cardiac SGLT1 protein occurs at a post-transcriptional level. Co-immunoprecipitation of SGLT1 with ubiquitin was reduced in diabetic human and rat hearts, which indicates that SGLT1 degradation is impaired. The E3 ubiquitin ligase Nedd4-2 is responsible for ubiquitination of several cardiac membrane transporters. Pharmacological inhibition of Nedd4-2 with Heclin in HL-1 cells resulted in significantly higher SGLT1 protein levels, suggesting that Nedd4-2 also mediates SGLT1 degradation. Nedd4-2 is inactivated upon phosphorylation by several kinases, including the serum and glucocorticoid-regulated kinase-1 (SGK1). Using western blotting, we found increased Nedd4-2 phosphorylation in hearts from humans and rats with T2D compared to their non-diabetic counterparts. Moreover, hearts from T2D patients exhibited higher levels of SGK1 and its activated phosphorylated form compared to their non-diabetic counterparts.

Conclusion/New Hypothesis:
The increase in SGLT1 protein in the diabetic heart is regulated post-transcriptionally by an impairment in Nedd4-2 dependent ubiquitination and degradation of SGLT1.
Abstract P1166: O-GlcNAcylation Regulates Protein Synthesis In Cardiomyocytes Through Effects On The Translational Regulator Eif2α
Authors: Papanicolaou Kyriakos , Jung Jessica , Zhang Wenxi , Ashok Deepthi , Foster D.Brian , O’Rourke Brian , Zachara Natasha
Published: October 2023

The regulated and reversible modification of Ser/Thr residues by O-linked N-Acetylglucosamine (O-GlcNAc) is termed O-GlcNAcylation. O-GlcNAc cycling on and off proteins is regulated by two conserved enzymes, the ‘writer’ O-GlcNAc Transferase (OGT) and the ‘eraser’ O-GlcNAcase (OGA). Crucially, O-GlcNAcylation is implicated in nutrient sensing, cell growth and stress adaptation. Despite these significant roles, the precise pathways impacted by O-GlcNAcylation and what key substrate proteins are involved remain incompletely understood. A hallmark of cardiomyocyte hypertrophy is the rapid production of new proteins that assemble into the contractile machinery of the growing myocyte. eIF2α is a rate-limiting factor in translation initiation. Interestingly, phosphorylation of eIF2α causes the arrest of general mRNA translation and favors the translation of stress-adaptive mRNAs. In the present work we investigated whether altering O-GlcNAcylation affects nascent protein synthesis (NPS) in hypertrophic cardiomyocytes. NPS was significantly elevated by the agonist phenylephrine, and this response was suppressed by OSMI-1 (OGT inhibitor). Moreover, we found that OGT-inhibited cells had increased phosphorylation of eIF2α. Additionally, we found increased protein levels of the stress adaptive factors Atf4 and Chop, whose mRNA translation is favored when eIF2α is phosphorylated. ISRIB, a small molecule designed to suppress the effects of phospho-eIF2α in altering translation, significantly prevented OSMI-1-induced accumulation of Atf4. Furthermore, we used metabolic labeling of glycans in cells, coupled with alkyne-azide ‘click’ chemistry and streptavidin pull-down, to show that eIF2α can be O-GlcNAcylated in cardiomyocytes. Furthermore, protein enrichment followed by mass spectrometry identified potential sites of O-GlcNAcylation on eIF2α. Taken together, these results highlight a novel role of O-GlcNAcylation on cardiomyocyte protein synthesis through the regulation of eIF2α. Future investigations will characterize the upstream mechanisms that promote eIF2α phosphorylation in OGT-inhibited cells and investigate the implications of direct eIF2α O-GlcNAcylation on hypertrophic protein synthesis in cardiomyocytes.
Abstract P1176: What Is The Nature Of The Hypertrophic Response Contributing To Pregnancy-induced Cardiac Growth And Its Reversal?
Authors: Collins Helen E , Schulman Emily
Published: October 2023

During pregnancy, the maternal heart undergoes physiological cardiac growth, reversing following parturition; however, the contributing underlying processes are largely unknown. Changes in cardiac metabolism appear to be associated with cardiac growth through a reduction in glucose catabolism. We have also shown that several cardiac metabolic transcripts, proteins, and liver-derived metabolites that are elevated during pregnancy are associated with cardiac growth. However, the nature of the hypertrophic response observed in the maternal heart is unclear. Therefore, this study aimed to examine the metabolic and hypertrophic changes associated with pregnancy-induced cardiac growth and reversal. Studies revealed that both heart weight (HW: TL;
p
< 0.0001) and liver weight (LIV: TL;
p
<0.0001) were highest in lactating 1-week post-birth (PB) mice versus mid-pregnant (8 d), late-pregnant (LP; 16 d), and non-pregnant (NP) 12-week-old FVB/NJ mice. Of note, we also found that HW: TL (
p
< 0.01) and LIV: TL (
p
< 0.0001) were significantly reduced in non-lactating 1-week post-birth mice (PBNL) compared with PB mice. However, the changes in cardiac size observed during pregnancy and post-birth were not associated with changes in either cardiomyocyte cross-sectional area or cardiac fluid content. Hearts and livers remained significantly larger, irrespective of lactation status, following parturition through 6 weeks post birth (PB) versus NP. Consistent with these changes, we found significant reductions in Pdk4 protein between 1- and 3-week PB and PBNL groups that were reversed by six weeks postpartum and a substantial increase in Pdk4 expression at six weeks postpartum, respectively. Interestingly, we see a sustained increase in cardiac Bdh1 expression that persisted at six weeks postpartum, which could contribute to the sustained increases in HW: TL versus NP. Changes in lactation status and metabolism are likely contributing to similar patterns of growth seen in the heart and liver during and following pregnancy. The maternal hypertrophic response is considered eccentric due to the absence of changes in myocyte cross-sectional area and cardiac fluid content.
Abstract P1182: The Genetic Dissection Of
Rpl3l
As A Causal Gene For Neonatal Dilated Cardiomyopathy And Left Ventricular Noncompaction
Authors: Orgil Buyan-Ochir , Gu Qingqing , Martinez Hugo , Bajpai Akhilesh , Absi Mohammed , Ryan Kaitlin , Alberson Neely , Lu Lu , Towbin Jeffrey A , Purevjav Enkhsaikhan
Published: October 2023

Background:
Cardiomyopathies are responsible for nearly 10% of cardiac deaths in neonates. Recently, biallelic variants in ribosomal protein L3-like (
RPL3L
, NM_005061.2) have been reported in neonates with dilated cardiomyopathy (DCM) and heart failure (HF). This study is aimed to study
RPL3L
induced genetic pathways and mechanisms involved in the development of DCM.

Methods:
Whole exome and RNA sequencing, histology, immunohistochemistry, and Western blotting were performed in a human explanted heart. Genetic correlation, expression quantitative trait loci (eQTL) mapping and functional analysis were performed using cardiac gene expression data from the patient as well as human and murine genetic reference populations (GRPs).

Results:
Two-month-old patient with DCM and HF carrying compound heterozygous
RPL3L
variants, p.A51T and p.V231F, has been managed on an INTERMACS profile 2 with continuous-flow ventricular assist device and successfully bridged to heart transplantation at the age of 4 months. Morphology of the explanted heart displayed apical deep intertrabecular recesses consistent with left ventricular noncompaction (LVNC). Histology revealed interstitial fibrosis and myocardial hypertrophy. No signs of myocarditis and glycogen or mucopolysaccharides deposits were detected. Immunohistochemistry revealed abnormal clusters of RPL3L protein in the cytosol of cardiomyocytes and disruption of F-actin, Z-disk proteins, MYL4 and dystrophin was evident. In a mouse heart, the
Rpl3l
level was of 33.45 RPKM, while levels of
RPL3L
appeared to be 32 RPKM in a human heart compared to its mean expression in other adult tissues of 2.54 (mouse) and 2.36 (human). Systems genetics analysis identified high expression values ranged from 11.31 to 12.16 across murine GRPs of BXD mice with the ~1.8-fold difference. Pathways such as “thermogenesis”, “diabetic cardiomyopathy” and “DCM” significantly associated with
RPL3L. e
QTL mapping suggested
Myl4
(Chr 11) and
Sdha
(Chr 13) as the upstream regulators of
Rpl3l
.

Conclusions:
Compound
RPL3L
heterozygosity is causal for neonatal DCM and LVNC. The
RPL3L
is highly expressed in the heart tissue of humans and mice. Genes,
Myl4
and
Sdha,
are strong candidates that regulate expression of
Rpl3l
in murine heart.
Abstract P1184: Spatiotemporal Proteomics Approach To Discern New Features Of Carfilzomib-mediated Cardiotoxicity
Authors: Currie Jordan , Manda Vyshnavi , Hidalgo Veronica , Ludwig Robert W , Lam Maggie P , Lau Edward
Published: October 2023

Background:
Proteasome inhibitors including carfilzomib have led to remarkable improvement in the survival of multiple myeloma patients, but are associated with cardiovascular adverse events including heart failure. A better understanding of the mechanism of toxicity is needed to improve oncogenic and cardiovascular outcomes.

Rationale:
Proteasome inhibition is known to impair the degradation rates as well as the subcellular distribution of proteins, processes that are not fully reflected by differential gene expression analysis. We hypothesize that cardiomyocyte proteins show impaired spatiotemporal distributions upon carfilzomib exposure, and that these changes constitute a crucial under-examined aspect of cardiotoxicity mechanism.

Approach:
To test this hypothesis, we developed a new mass spectrometry-based proteomics strategy that can simultaneously trace the turnover kinetics and subcellular localizations of proteins in cardiac cells.

Results:
In preliminary work, we applied this method to human iPSC-derived cardiomyocytes under 0.5 μM carfilzomib vs. control (n=2). We quantified the localization and turnover rates of >3,500 proteins in control and carfilzomib-treated cells. Unexpectedly, no significant difference was identified in the global rate of protein turnover between control and carfilzomib treated cells (p = 0.24 Wilcoxon-Mann-Whitney test), where the median protein half-lives of of control and carfilzomib treated cells were 31.0 vs. 30.7 hours, respectively. However, when we used a T-augmented Gaussian mixture model to predict protein localizations and assess compartment-specific turnover changes, we observed a significant reduction in turnover in the nuclear fraction, and a concomitant increase in that of proteasomal fraction under carfilzomib. The data also revealed a change in assigned localization in many myosin-binding proteins toward lysosomal co-sedimenting compartments, consistent with sarcomeric disarray and degradation in carfilzomib-exposed cardiomyocytes. Finally, we show a decrease in turnover of both fatty acid and glucose metabolism associated proteins. This method has potential for discovering unique molecular features of proteasome inhibitor mediated cardiotoxicity.
Abstract P2019: Diastolic Dysfunction In Alzheimer's Disease Is Associated With aβ- Amyloid Aggregate Formation And Mitochondrial Dysfunction
Authors: Aishwarya Richa , Abdullah Chowdhury S. , Remex Naznin Sultana , Nitu Sadia , Stokes Karen , Bhuiyan Md Shenuarin
Published: October 2023

Background:
Alzheimer's disease (AD) is a neurodegenerative disease with memory impairment caused by an accumulation of toxic protein aggregates (Aβ- amyloid plaques and tau-derived neurofibrillary tangles). Several studies reported cardiovascular abnormalities with diastolic dysfunction in AD patients; however, the direct mechanism of cardiac dysfunction in AD patients remains unexplored. Therefore, we investigated the pathophysiological mechanism of cardiac dysfunction in the mice model of AD.

Hypothesis:
Two most commonly used mice models of AD was thoroughly evaluated for cardiac dysfunction, histopathology, and biochemical changes over aging.

Methods and Result:
To determine the mechanism of AD-associated cardiac dysfunction, we used two different AD mice models: double transgenic mice expressing a chimeric mouse/human amyloid precursor protein and a mutant human presenilin 1 (APP/PS1 Tg), and transgenic mice that is homozygous for a
Psen1
mutation and the co-injected APPSwe and tauP301L transgenes (3xTg-AD). Cardiac function measurement using non-invasive echocardiography in aged Ntg and AD mice models showed no difference in systolic cardiac functional parameters. However, diastolic function assessment using Doppler echocardiographic measurements showed ventricular diastolic dysfunction with reduced diastolic filling in APP/PS1 Tg and 3xTg-AD mice. Western blot analysis showed Aβ- amyloid protein expression in APP/PS1 Tg hearts. Immunohistochemical and immunofluorescence staining of the APP/PS1 Tg heart sections further confirmed the Aβ- amyloid protein expression and Aβ- amyloid aggregate formation in the cardiomyocytes. Further evaluation of APP/PS1 Tg hearts demonstrated increased collagen deposition (Picro-Sirius red staining) and increased cardiomyocyte cross-sectional area. Interestingly, APP/PS1 Tg hearts also displayed mitochondrial dysfunction with decreased mitochondrial bioenergetics and significantly reduced mitochondrial function regulatory parameters.

Conclusion:
Overall, we found diastolic dysfunction development with Aβ- amyloid aggregate formation, fibrotic remodeling, increased cardiomyocyte cross-sectional area, and mitochondrial dysfunction in the AD mice hearts.
Abstract P2027: Chronic β-Adrenergic Receptor Stimulation Exacerbates Left Ventricular Dilation And Myocardial Cell Death In Rgs2 Knockout Mice
Authors: Dahlen Shelby
Published: October 2023

Sustained sympathoexciation and increased circulating catecholamine level are hallmarks of congestive heart failure. Elevated catecholamine level promotes a sustained β -adrenergic receptor (βAR) stimulation in the myocardium, altering the balance between cell death and survival via intracellular signaling downstream of G
s
- and G
i/o
-class G proteins. Previously, we and others reported that moderation of Gα
i
activity by regulators of G protein signaling (RGS) 2 and 5 separately is key to maintaining normal ventricular rhythm. Here, using single isogenic wild type (WT),

Rgs2
-/-
,

Rgs5
-/-

, and
Rgs2/5
double knockout (dbKO) adult male mice with or without systemic isoproterenol infusion, we determined whether a compensatory relationship between RGS2 and RGS5 is cardioprotective and promotes myocardial cell survival during chronic sympathoexcitation. We found that basal heart weight-to-tibia-length (HW/TL) ratio of both
Rgs2
and dbKO mice was elevated relative to WT control. Echocardiography revealed left ventricular dilation in all three KO genotypes, as evidenced by increased left ventricular internal diameter and volume. Both

Rgs2
-/-

and
Rgs5-/-
mice displayed reduced ejection fraction and fractional shortening. To explore the mechanism mediating the LV dilation, we challenged the mice with chronic βAR stimulation by systemic isoproterenol infusion (ISO, 30 mg/kg/day) for 3 or 14 days via subcutaneous osmotic mini-pumps. ISO infusion substantially increased HW/TL ratio in WT mice after day 3, while HW/TL ratio in

Rgs2
-/-

and dbKO mice was increased with exacerbated LV dilation. After 14-day ISO infusion, only dbKO mice displayed increased lung weight/TL ratio, indicating a congestive heart failure. Additionally, myocardial tissue from saline treated dbKO mice had a larger number of TUNEL
+
cells that was further increased by ISO infusion, with extensive interstitial fibrosis as visualized with Masson trichrome staining, and increased expression of heart failure biomarkers
Mybpc3
and
Tnni3
. Taken together, we conclude that the loss of
R
gs2,
more-so than
Rgs5,
predisposes to myocardial cell death and rapid progression to dilated cardiomyopathy and heart failure during sustained sympathoexciation and β-adrenergic stimulation.
Abstract P2031: Myofibroblast-specific Smad7 Attenuates Fibrosis Of The Pressure-overloaded Heart By Inhibiting Mmp2-mediated Collagen Denaturation And By Restraining Macrophage Activation.
Authors: Humeres Claudio , Hernandez-Velasco Silvia C , Hanna Anis , Tuleta Izabela , Venugopal Harikrishnan , Kubota Akihiko , Frangogiannis Nikolaos
Published: October 2023

Expansion and activation of cardiac fibroblasts contributes to adverse remodeling, fibrosis and dysfunction in the pressure-overloaded heart. Although early fibroblast TGF-β/Smad3 activation protects the pressure overloaded heart by preserving the matrix, sustained TGF-β activation may be deleterious, accentuating fibrosis and dysfunction. We hypothesized that induction of the endogenous TGF-β inhibitory Smad, Smad7, may attenuate fibroblast activation and deposition of collagen, limiting myocardial fibrosis and dysfunction in the pressure-overloaded heart. In a mouse model of pressure overload induced through transverse aortic constriction (TAC), Smad7 was upregulated in cardiac fibroblasts and myofibroblasts. Mice with myofibroblast-specific loss of Smad7 had increased mortality, systolic dysfunction, dilative remodeling and accelerated diastolic dysfunction in response to TAC. Moreover, myofibroblast-specific Smad7 loss hearts had higher collagen deposition and denaturation, as well as increased deposition of the fibrogenic protein periostin. Secretomic analysis showed that fibroblast Smad7 loss accentuates secretion of matrix metalloproteinase-2 (MMP2), extracellular matrix proteins and structural collagens. In vitro, the antifibrotic effects of Smad7 on fibroblast-induced collagen denaturation and pad contraction were partly mediated via MMP2 downregulation. Ingenuity pathway analysis predicted the fibrogenic matricellular protein CCN2/CTGF to be activated in Smad7 null fibroblasts. Surprisingly, myofibroblast-specific Smad7 loss increased myocardial macrophage infiltration in the pressure-overloaded heart. In vitro experiments suggested paracrine effects of Smad7 KO fibroblasts on macrophage proliferation and fibrogenic activation that involved the fibroblast-secreted mediators CD5L, SPARC, CTGF and TGFBI. In summary, the anti-fibrotic effects of Smad7 in the pressure-overloaded heart involve not only reduction in collagen deposition, but also suppression of MMP2-mediated matrix denaturation and paracrine effects inducing fibrogenic activation of macrophages.
Abstract P2038: Role Of The Microtubule Network In Right Ventricle Passive Viscoelasticity With Pulmonary Hypertension Development
Authors: LeBar Kristen , Chicco Adam J , Wang Zhijie
Published: October 2023

Microtubule (MT) polymerization is a key contributor to myocardium viscoelasticity, which describes elastic and viscous resistance under stress and impacts the contractile function. MT de- or hyper-polymerization has been shown to decrease or increase healthy cardiomyocyte viscoelasticity, but the responses of hypertrophied cells to these modifications are unknown. Moreover, the ventricular free wall is a complex tissue composed of myofibers, collagen and vasculature, and it exhibits anisotropic mechanical behavior. To fully understand the role of MT in the tissue-level anisotropic viscoelasticity of myocardium, we investigated the responses of healthy and failing rat right ventricles (RV) to alterations in MT network. All procedures were approved by Colorado State University IACUC. Adult male rats were treated with monocrotaline to induce pulmonary hypertension and RV failure. After euthanasia, the RV free wall underwent equibiaxial stress relaxation in intact state (baseline) and under the treatment of colchicine and deuterium oxide (D
2
O) to remove and enhance MT network, respectively. Viscoelastic parameters were quantified as previously described. As expected, the diseased RV had greater viscoelasticity than the healthy RV. Depolymerization of MT in healthy RVs reduced tissue viscosity (relaxation rate) and elasticity (relaxation modulus), and the changes were more significant in the longitudinal (outflow tract) direction. MT hyper-polymerization increased tissue elasticity and decreased viscosity, with the changes stronger in the circumferential direction. In diseased RVs, MT depolymerization reduced viscosity in both directions and elasticity only in the circumferential direction. However, MT hyper-polymerization unexpectedly decreased tissue elasticity and tended to decrease viscosity, as well. The altered MT polymerization dynamics after D
2
O treatment in a fibrotic environment may explain these heterogeneous responses This is the first report of distinct impacts of MT polymerization on the tissue mechanical behavior between the hypertrophied and healthy myocardium. The exact molecular mechanisms involving MT polymerization dynamics and the interactions with collagen await further investigation.
Abstract P2045: Regulation Of Atrial Natriuretic Peptide Secretion By Palmitoylation And Modulation Of The Rab3a GTPase Cycle In Cardiac Myocytes
Authors: Essandoh Kobina , Subramani Arasakumar , Teuber James , Brody Matthew
Published: October 2023

Despite the clinical relevance to cardiovascular disease, molecular regulation of atrial natriuretic peptide (ANP) release by cardiomyocytes remains ill-defined. We uncovered novel functions for Rab3gap1 and modulation of Rab3a nucleotide cycling in membrane fusion and atrial natriuretic peptide (ANP) release in cardiomyocytes. We generated transgenic mice overexpressing the Golgi-localized S-acyltransferase, zDHHC9, and identified Rab3gap1 as a novel cardiac substrate of zDHHC9 by S-acyl proteomics. Rab3gap1 palmitoylation and retention at the cardiomyocyte Golgi, Rab3a-GTP levels, and formation of Rab3a-positive post-Golgi vesicles were all enhanced in zDHHC9 overexpressing hearts as were Rab3gap1, Rab3a, and ANP atrial protein levels but circulating ANP levels were reduced, suggesting a defect in ANP secretion. Notably, treatment of cardiomyocytes with the secretagogue phenylephrine (PE) promoted substantial ANP release as expected, but also enhanced Rab3gap1 palmitoylation and Rab3a GTP-loading and peripheral vesicle formation, whereas knockdown of zDHHC9 repressed these PE-induced effects on Rab3gap1 and Rab3a but further enhanced PE-stimulated ANP secretion, suggesting zDHHC9-mediated palmitoylation of Rab3gap1 serves as an intrinsic mechanism to limit ANP release. Consistent with a role for Rab3a nucleotide exchange in modulation of ANP release, overexpression of Rab3gap1 in cardiomyocytes promoted a profound increase in ANP release whereas a GAP-deficient mutant of Rab3gap1 (Rab3gap1
R728A
) was resistant to this effect. Collectively, these data suggest disruption of Rab3a nucleotide cycling perturbs secretory vesicle membrane fusion and ANP release in cardiac myocytes and establish critical roles for Rab3a activity and its modulation by zDHHC9-mediated palmitoylation of Rab3gap1 in governing cardiac homeostasis and endocrine signaling.