bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract P1128: Myocardial Delivery Of Modified Mrna For Exosomal Protein Induces Cardiac Regeneration
Authors: Magadum Ajit , Mallaredy Vandana , Roy Rajika , Wang Chunlin , Joladarashi Darukeshwara , Gurrala Charan Thej , Cheng Zhongjian , Truongcao May , Lucchese Anna Maria , Benedict Cindy , Rigaud Vagner O , Khan Mohsin , Kishore Raj
Published: November 2022

Permanent loss of cardiomyocytes (CM) after myocardial infarction (MI) and limited cardiac regenerative capacity leads to heart failure. Recent studies from zebrafish, newt, and even 1-day old neonatal mice showed that they could regenerate their heart through inducing existing CM proliferation. Attempts have been made to transiently reconstitute embryonic signaling in adult hearts, including overexpression of cell cycle activating genes with limited success. iPSC-derived extracellular vesicles (EV)/exosomes have been shown to improve cardiac function and some degree of CM renewal. However, the iPSC-EVs-mediated cardiac regeneration mechanism remains unclear and largely pertains to microRNAs and other RNAs, but not the proteins. Our hypothesis is the myocardial delivery of iPSC-EV-specific protein improves cardiac function and remodeling post-MI by activating pro-proliferative and anti-oxidative stress molecular pathways. Our preliminary studies showed that hiPSC-EVs induced the CM cell cycle in mice post-MI, and by employing a proteomic approach, we found a novel protein exclusively expressed in iPSC-EVs. The overexpression of hiPSC-EV enriched protein in the form of modRNA (modified mRNA) induces a robust CM cell cycle in rat neonatal CMs and in heart post-MI. By employing cardiomyocyte specific MADM (Mosaic Analysis of Double Markers) mice we showed that EV-protein induced robust cardiomyocyte division post-MI. This increase in the CMs proliferation by the modRNA was associated with reduced scar size, improved cardiac function, and mice survival 28 days post-MI. Moreover, we show that the YAP, a master regulator of CM proliferation and cardiac regeneration, binds to the promoter of the protein and induces its expression. Furthermore, using the siRNA and modRNA (inhibition and over-expression respectively) approach, we found that the protein-induced Yap1-β-catenin molecular pathway stimulates CM proliferation. Furthermore, the overexpression protein inhibited the oxidative stress or ROS analyzed by HPLC and DNA damage response post-MI. As a result of it, we have seen a significant reduction in CM apoptosis post-MI. Taken together we see the myocardial injection of iPSC-EV specific protein through a highly therapeutic modRNA tool improve cardiac function by inducing CMs proliferation, inhibiting oxidative stress, and reactivating cardiac regeneration post-injury.
Abstract P2001: Vessel Normalization By Targeting Endothelial Cell Plasticity To Improve Cardiac Repair After Myocardial Infarction
Authors: Huang Menggui , Yang Fan , Zhang Duo , Lin Maohuan , Pei Liming , Mitchell Michael J. , Rader Daniel J , Fan Yi , Gong Yanqing A
Published: November 2022

Ischemic heart disease is a leading cause of death in the world. Formation of new blood vessels is fundamental to cardiac reperfusion and repair after myocardial infarction (MI), and therapeutic promotion of angiogenesis, therefore, has emerged as a promising strategy for treating ischemic heart diseases. However, the therapeutic efficacy is small and transient in most of pro-angiogenic therapy. Here, we characterize unexpectedly prominent vessel aberrancy, albeit a robust angiogenesis, in MI-associated cardiac tissue by 3-dementional light sheet fluorescence imaging. Our single-cell transcriptome analysis of mouse MI tissues with
Cdh5-Cre
;
Rosa-LSL-tdTomato
-mediated endothelial lineage tracing identifies a robust EC plasticity to acquire mesenchymal gene signature in MI-associated ECs, peaked at 1 week and partially retained at 4 and 8 weeks after MI. Moreover, cardiac ECs undergo transcriptional activation to drive mesenchymal phenotypes including over-proliferation, migration and increased permeability, which lead to vessel abnormality after exposure to
in vitro
ischemic microenvironment. Furthermore, our single-cell and bulk RNA-seq analysis uncovers a PDGF/NF-κB/HIF-1α axis that induces Snail expression and mesenchymal phenotypes in ECs under hypoxia, culminating in aberrant vascularization. Notably, EC-specific knockout of PDGFR-β abrogates mesenchymal gene expression in ECs, attenuates vascular abnormalities in the infarcted tissue, and improves cardiac repair after MI. Interestingly, our single-nuclei analysis uncovers that genetic ablation of PDGFR-β in ECs temporally reprograms cardiomyocyte metabolism and enhances myocyte survival and proliferation. Pharmacological PDGFR inhibition or nanoparticle-based targeted PDGFR-β siRNA delivery normalizes vasculature and improves cardiac function recovery after MI. These findings illustrate a mechanism controlling aberrant neovascularization after ischemia, and suggest that targeting PDGF/Snail-mediated endothelial plasticity may offer exciting opportunities for normalizing vasculature and treating ischemic heart disease.
Abstract P2002: Mst1 Suppresses Pressure Overload-induced Cardiac Hypertrophy But Reduces Cardiac Function Through PERK Phosphorylation
Authors: Mukai Risa , Usui Soichiro , Zhai Peiyong , Nakada Yasuki , Sadoshima Junichi
Published: November 2022

Cardiac hypertrophy in response to hemodynamic overload is initially an adaptive mechanism to maintain normal cardiac function by reducing left ventricular (LV) wall stress. Conversely, insufficient hypertrophy could lead to elevated wall stress, causing increased cell death and cardiac dysfunction. The Hippo pathway plays an important role in determining organ size. Mammalian sterile 20-like kinase 1 (Mst1) is the key serine-threonine kinase in the Hippo pathway, controlling cell death, proliferation, and differentiation. Mice with cardiac-specific overexpression of Mst1 (Tg-Mst1) fail to develop compensatory hypertrophy despite LV dysfunction and dilation at baseline. To elucidate the mechanism through which Mst1 inhibits cardiac hypertrophy, pressure overload (PO) was imposed on WT and Tg-Mst1 mice by transverse aortic constriction (TAC). TAC-induced increases in LV weight/body weight were significantly suppressed in Tg-Mst1 mice (5.8 vs WT 5.1, p<0.05). LV ejection fraction (EF) after TAC was lower in Tg-Mst1 mice than in WT mice (40% vs WT 60%, p<0.01), consistent with the notion that Mst1 promotes PO-induced systolic dysfunction in part by inhibiting compensatory hypertrophy. Mst1 interacted with and induced phosphorylation of protein kinase RNA-like endoplasmic reticulum kinase (PERK). We generated knock-in mice expressing phosphorylation-resistant PERK (PERK-KI) and performed TAC on WT and PERK-KI mice. LVEF was preserved in PERK-KI mice compared to in WT mice (67% vs WT 46%, p<0.05) after 8 weeks of TAC. PO-induced increases in lung congestion were attenuated in PERK-KI mice compared to in WT mice (10 vs WT 11.5, p<0.05). Despite the improvement in cardiac function, cardiac hypertrophy was not attenuated in PERK-KI mice. These results suggest that phosphorylation of PERK by Mst1 is detrimental for the heart during PO, in part due to suppression of compensatory hypertrophy.
Abstract P2003: ER-resident UFBP1 Maintains ER Homeostasis And Represses Cardiac Inflammation To Constrain Pathological Cardiac Remodeling
Authors: Li Jie , Ma Wenxia , Zhang Aizhen , Ayala Juan , Li Honglin , Su Huabo
Published: November 2022

Ufmylation is a novel protein modification by which protein substrates are covalently modified by the ubiquitin-like protein, UFM1 (ubiquitin-fold modifier 1) via a UFM1-specific E1-E2-E3 enzymatic cascade. Through its ability to regulate protein function, ufmylation participates in diverse pathophysiological events and is implicated in various human diseases. We recently reported that ufmylation is dysregulated by cardiomyopathy in both human and mouse hearts. Inhibition of ufmylation by targeting the E3 ligase, UFL1, in the heart evokes ER stress, leading to dilated cardiomyopathy and heart failure under resting and stress conditions, demonstrating its critical role in maintaining ER and cardiac homeostasis. However, how ufmylation regulates ER and cardiac function remain unclear. Here, we report that UFBP1 (UFM1 binding protein 1), an ER-resident ufmylation target, is a crucial downstream effector of ufmylation in the heart. UFBP1 is downregulated in UFL1-deficient hearts but upregulated in pressure overload hearts. Condition knockout (KO) of UFBP1 in mouse heart caused dilated cardiomyopathy during aging and exacerbated pressure overload-induced pathological cardiac remodeling. Of note, the timeline of disease progression in UFBP1 KO mice closely aligned with that of UFL1 KO mice. Transcriptomics analysis revealed the involvement of ER stress signaling and unexpectedly, inflammation in the pathogenesis. Biochemical analyses confirmed the pronounced ER stress, increased cytokine expression, and remarkable infiltration of inflammatory cells into the myocardium, which preceded the severe cardiac phenotype. Alleviation of ER stress via TUDCA administration mitigated UFBP1KO-induced cardiac dysfunction, suggesting a causal role of ER stress in the pathogenesis. Re-expression of wild-type UFBP1, but not its ER-binding–deficient mutant, in UFBP1 KO hearts rescued the pathological cardiac phenotype. UFPB1 expression was stabilized by UFL1, and depletion of UFBP1 in cardiomyocytes (CMs) induced ER stress and promoted cell death, recapitulating most aspects of UFL1-deficient CMs. Collectively, our data suggest that ER-resident UFBP1 represses ER stress and cardiac inflammation to constrain pathological cardiac remodeling.
Abstract P2007: Bmal1 Drives Postnatal Cardiac Hypertrophy Via Circadian Chromatin Remodeling Of The Pro-hypertrophic Gene
Sik1
Authors: Arrieta Adrian , Reese Anna , Wong David , Chapski Douglas J , Rosa-Garrido Manuel , Zhu Ashley , Vondriska Thomas M
Published: November 2022

Background:
During neonatal cardiac development, myocytes integrate cues such as changes in blood pressure and circulating hormones to execute a temporal genetic response that drives physiological cardiac hypertrophy. Chromatin remodeling precedes the adult heart’s response to growth stimuli, yet the temporal nature of neonatal chromatin remodeling is unclear. In other tissues, genes regulated by the master circadian rhythm factor BMAL1 display circadian oscillations in histone acetylation and nucleosome and RNAP II occupancy. We hypothesized that BMAL1 regulates temporal chromatin remodeling and expression of genes critical for neonatal cardiac hypertrophy.

Methods:
BMAL1 chromatin immunoprecipitation sequencing (ChIP-Seq) and assay for transposase-accessible chromatin and sequencing (ATAC-Seq) data from murine hearts were analyzed to identify genes at which BMAL1 may drive chromatin remodeling. To interrogate myocyte time-of-day dependent response to growth stimuli, neonatal rat ventricular myocytes (NRVM) were subjected to serum shock synchronization to drive oscillatory BMAL1 expression, followed by treatment with the growth stimulus phenylephrine (PE) at the peak and trough of BMAL1 expression. BMAL1 was knocked down via siRNA and hypertrophy was assessed by measurement of cell size and of RT-PCR of fetal genes (Nppa and Nppb).

Results:
BMAL1 ChIP-Seq data revealed myocyte-specific BMAL1 localization to regions exhibiting an open chromatin signature based on ATAC-seq, including the pro-hypertrophic gene salt-inducible kinase 1 (
Sik1
). Serum shock induced oscillations of BMAL1 and histone H3.3: administration of PE at the peak of BMAL1 expression induced a 30% increase in cell size and 2-fold increase in fetal gene expression. PE given at the trough of BMAL1 expression affected neither cell size nor fetal genes, demonstrating a critical role for the circadian clock in myocyte growth. BMAL1 knockdown: decreased expression of its known target and partner clock gene,
Per2
, as well as expression of
Sik1, Nppa
, and
Nppb
; decreased myocyte size by 30%; and increased expression of histone H3.3.

Conclusions:
These data indicate that BMAL1 temporally regulates myocyte growth by modulating histone stoichiometry and expression of
Sik1
.
Abstract P2013: Transcription Fidelity Dysregulation In Heart Failure Pathogenesis
Authors: Luo Weijia , Chen Zhishi , Yu Wei , Youker Keith , Chang Jiang
Published: November 2022

Using deep sequencing combined with computational biology, we found that the failing human hearts (n=3) contain a significant amount of aberrant transcripts arising from widespread spurious intragenic transcription initiations. The same phenomenon was detected in failing mouse hearts (n=3) induced by pressure overload and ischemia injury. Therefore, we hypothesize that spurious transcripts accumulation is a causative factor promoting heart failure (HF). The SET domain containing 2 (Setd2), an H3K36 tri-methylase, is a key regulator for transcription fidelity. The Setd2 modified H3K36me3 prevents RNA polymerase II (Pol II) entry from the gene body and the subsequent cryptic transcription initiation. In the same cohort of failing human and mouse hearts, we found that the SETD2 and H3K36me3 levels were concurrently reduced. To further test our hypothesis, we generated cardiac-specific Setd2-Knockout mice. The mice (n≥8) developed a typical HF progression phenotype: initial compensatory hypertrophy followed by a gradually deteriorating HF with chamber dilation. Transcriptome analysis revealed an accumulation of spurious transcripts in pre-hypertrophic Setd2
-/-
cardiomyocytes (n=3). New intragenic transcription initiation sites were identified by DECAP-seq in the knockout (n=3), confirming that the increased aberrant transcripts resulted from spurious Pol II entry. Using ribosome footprinting assay (n=3) to map the translation initiation sites, we found that the aberrant transcripts were partially translated into micro peptides in the Setd2
-/-
heart, suggesting that the accumulated aberrant transcripts and micro peptides together promote cardiac hypertrophy. Finally, we developed transgenic mice overexpressing human SETD2 (Tg-MHC-SETD2) in the heart. SETD2 overexpression repressed TAC-induced accumulation of spurious transcripts in cardiomyocytes (n=3). The transgenic mice (n≥6) were resistant to pressure overload stress with less cardiac hypertrophy and improved cardiac contractility. Together, we report that dysregulated transcription fidelity causes adverse aberrant transcripts accumulation leading to cardiac hypertrophy and heart failure. This new mechanism provides potential new therapeutic targets for HF.
Abstract P2014: Chitinase-3-like-1 Is A Neutrophil Secreted Factor That Antagonizes Replacement Fibrosis And Exacerbates Cardiac Dysfunction After Myocardial Infarction.
Authors: Stephan Jonah , Gu Zhen , Taylor Zoubul , Dassanayaka Sujith , Brainard Robert , Jones Steven P , Wysoczynski Marcin
Published: November 2022

Patient studies show that chitinase 3-like 1 (CHI3L1) is a novel biomarker of coronary artery disease (CAD). Furthermore, CHI3L1 plasma level is a strong predictor of all-cause of mortality in patients with CAD, and is a strong indicator of heart failure progression. Studies from our lab revealed that CHI3L1 is elevated in hearts after MI, and systemic administration of CHI3L1 in mice exacerbated post-MI fibrosis and left ventricular dysfunction. The goal of the current study was to delineate the mechanism of CHI3L1 secretion after MI and test the impact of CHI3L1 on post-MI replacement fibrosis. We found that CHI3L1 levels were rapidly increased in infarcted regions of LV at 2 d after MI and remained significantly elevated at 7 and 35 d (n=5-11/group, p<0.05). More detailed analysis revealed that neutrophils are the main source of CHI3L1. Protein CHI3L1 expression in neutrophils was 3-4 orders of magnitude higher compared with cardiac fibroblasts, macrophages, endothelial cells, and cardiomyocytes (n=3-5, p<0.0001). Next, we found that

Chil1
-/-

subjected to permanent coronary artery ligation showed decreased end-systolic and diastolic volumes and increased ejection fraction measured at 7 and 35 d with echocardiography (n=7-10/group, p<0.05 at 7 d; n=16-20/group, p<0.05 at 35 d). Histological analysis of hearts displayed no change in scar size but left ventricular expansion index was reduced and the infarcted wall thickness was increased in

Chil1
-/-

mice compared to their

Chil1
+/+

controls (n=16-18/group, p<0.01). We found no difference in cardiomyocyte cross-sectional area (WGA staining) and capillary density (isolectin B4 staining). These data provide proof of principle evidence that CHI3L1 is a neutrophil secreted factor that has an adverse effect on replacement fibrosis after MI. Future efforts will focus on the mechanism of actions of CHI3L1 and the identification of strategies to inhibit detrimental actions of CHI3L1 on replacement fibrosis and heart failure.
Abstract P2017: Proximity Proteomic Mapping Of Cav1.2 In Systolic Heart Failure
Authors: Kushner Jared , Liu Guoxia , Wan Elaine Y , Marx Steven
Published: November 2022

Introduction:
In heart failure (HF), there is dysregulation of Ca
V
1.2. This includes reduced expression, pathologic splice variation, and loss of β-adrenergic activation. We showed that adrenergic-induced inotropy requires PKA phosphorylation of Rad and disinhibition of Ca
V
1.2. We hypothesize that proximity labeling Ca
V
1.2 can illuminate important pathobiology in HF, such as whether loss of adrenergic activation follows absence of Rad from the channel nanodomain.

Methods:
Transgenic (TG) mice with cardiac expression of Ca
V
1.2 subunits fused to the engineered ascorbate peroxidase, APEX2 were made. HF was confirmed by echocardiography 3 months after coronary artery ligation. Multiplexed quantitative mass spectrometry was performed on homogenates of APEX2-catalyzed proximity labeled myocytes.

Results:
After surgery, 5 TG Ca
V
1.2 α
1C
-APEX and 5 β
2B
-APEX mice had FS less than 25%. The biotinylated proteins from the HF samples were compared to biotinylated proteins from 6 healthy α
1C
-APEX or β
2B
-APEX mice. More than 2000 proteins were quantified. We found significant enrichment of microtubule proteins (blue), the 26s proteasome (red), as well as key regulatory proteins such as Rad and PKA (green).

Conclusions:
We demonstrate that proximity proteomics has the power to probe deeply,
in vivo
, the molecular biology of HF at nanometer resolution. Attenuated adrenergic responses do not follow absence of PKA or Rad from proximity to Ca
V
1.2, and we identified a potential mechanism for loss of channel in HF—increased proteosomal degradation. Future studies will explore whether Rad is properly released from stimulated channels in HF as well as mechanisms of channel degradation.
Abstract P2032: Cardiac Nuclear Receptor Subfamily 4 Group A Member 2 Activates Cell Cycle Progression And Induces Heart Failure
Authors: Ashraf Sadia , Taegtmeyer Heinrich , Harmancey Romain
Published: November 2022

Transcription factors play a fundamental role in cardiovascular adaptation to stress. Nuclear receptor subfamily 4 group A member 2 (NR4A2; NURR1) is an immediate-early gene and transcription factor with a versatile role throughout many organs. In the adult mammalian heart and particularly in cardiac myocytes, NR4A2 is strongly up-regulated in response to beta-adrenergic stimulation. The physiological implications of this increase remain unknown. In this study, we aimed to interrogate the consequences of cardiac NR4A2 up-regulation under normal conditions and in response to pressure overload. In mice, tamoxifen-dependent, cardiomyocyte-restricted overexpression of NR4A2 led to left ventricular hypertrophy and dilation, heart failure, and death within 40 days. Chronic NR4A2 induction also precipitated cardiac decompensation during transverse aortic constriction (TAC)-induced pressure overload. Mechanistically, NR4A2 caused adult cardiac myocytes to return to a fetal-like phenotype, with a switch to glycolytic metabolism and disassembly of sarcomeric structures. NR4A2 also re-activated cell cycle progression and stimulated DNA replication and karyokinesis but failed to induce cytokinesis, thereby promoting polyploidization and multinucleation of cardiac myocytes. Activation of cell cycle checkpoints led to induction of an apoptotic response which ultimately resulted in excessive loss of cardiac myocytes and impaired left ventricular contractile function. In summary, myocyte-specific overexpression of NR4A2 in the postnatal mammalian heart results in increased cell cycle re-entry and DNA replication but does not result in cardiac myocyte division. Our findings expose a novel function for the nuclear receptor as a critical regulator in the self-renewal of the cardiac myocyte and heart regeneration.
Abstract P2038: Synthetic Membrane Stabilizers Reduce Serum Ck And Immune Cell Infiltrate In Neonatal Mdx Skeletal Muscle Prior To Overt Muscle Necrosis Onset In Vivo
Authors: Cohen Houda , Metzger Joseph M , Carlson McKayla
Published: November 2022

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive disease caused by a mutation in the dystrophin gene resulting in a complete loss of dystrophin from the striated muscle. This leads to the disruption of the dystrophin-associated glycoprotein complex and altered mechanical and signaling functions subsequently causing membrane instability, necrosis, and progressive muscle wasting. Immune cell infiltration is a prominent feature of the DMD pathophysiology and is strongly associated with disease severity. To date, no curative treatment is available for DMD. A logical therapeutic approach for DMD is to directly target the primary defect of DMD, sarcolemma instability, and thus prevent contraction-induced membrane damage. First in class synthetic triblock copolymer, poloxamer 188 (P188), is a muscle membrane interfacing and stabilizing molecule that has been shown to protect dystrophic myocardium and skeletal muscle from acute stress induced injury
in vivo
. Creatine kinase (CK) is a well-established biomarker of DMD that reflects sarcolemma damage, and serum levels have been shown to dramatically increase in mdx mice, but this has not been fully characterized during the early postnatal growth period prior to overt disease onset at 3 weeks. Under standard laboratory housing conditions for adult mdx mice (6-9 month old), we found that daily administration of P188 for two weeks did not significantly change serum CK levels or Evan’s blue dye uptake by diaphragm, heart or tibialis anterior muscles. In order to evaluate P188’s ability to protect dystrophin-deficient striated muscle prior to the overt disease, serum CK levels need to be characterized during this time period to identify the earliest muscle membrane damage event in mdx striated muscle and thus the earliest time point for therapeutic intervention. Results show that mdx serum CK levels are significantly higher than control serum levels 15 days after birth and continue to increase dramatically to 20 days after birth, and then CK remains high for the lifespan. Systemic delivery of P188 to neonatal mdx mice (12 days old) prior to CK peak significantly reduced serum CK levels and blunted immune cell infiltrate as compared to saline control mdx mice. These findings support the hypothesis that early intervention with membrane stabilizers has the potential to mitigate muscle damage and blunt the inflammatory response characteristic of DMD disease pathology. More work must be done to fully understand the early pathophysiological changes associated with dystrophin deficiency in order to implement novel therapeutic strategies including membrane stabilizers to enhance the standard of care for these patients and their families.
Abstract P2039: The Absence Of Sigmar1 Causes Increased Protein Aggregation And Aggravated Cardiac Dysfunction In R120g- αb-Crystallin Mouse
Authors: Aishwarya Richa , Abdullah Chowdhury S , Remex Naznin Sultana , Nitu Sadia S , Hartman Brandon , King Judy A , Bhuiyan Md. Shenuarin
Published: November 2022

Background:
αB-Crystallin (CryAB) is a molecular chaperone responsible for protecting intermediate filaments by maintaining the structural integrity of the sarcomere. Mutation in CryAB at Arg120Gly (CryAB
R120G
) causes the formation of toxic pre-amyloid oligomers and protein aggregation, contributing to cardiac dysfunction. In the event of proteotoxic insult, the chaperone proteins function to prevent the accumulation of protein aggregates. One of the multitasking chaperone proteins abundantly expressed in the heart and known to have molecular roles in the clearance of misfolded protein aggregates is Sigmar1. However, Sigmar1’s role in cardiac proteotoxicity and protein aggregation remains unknown.

Hypothesis:
We hypothesize that Sigmar1 plays a protective role in the degradation of protein aggregates in the heart. Here, we investigated the functional role of Sigmar1 in misfolded proteins aggregation-induced pathological cardiac remodeling and heart failure in CryAB
R120G
mice.

Methods and Result:
We measured the Sigmar1 expression level in CryAB
R120G
hearts and found that endogenous Sigmar1 protein and transcript levels are substantially reduced in CryAB
R120G
hearts. To test the functional role of Sigmar1 in a proteotoxic model of heart failure
in vivo
, we crossed the Sigmar1 knockout mouse with the CryAB
R120G
mouse to generate Sigmar1 ablated CryAB
R120G
mouse (CryAB
R120G
xSigmar1
-/-
). Echocardiographic analysis showed the age-dependent aggravation of cardiac dysfunction in the CryAB
R120G
xSigmar1
-/-
mice compared to CryAB
R120G
mice. Masson’s Trichrome staining also showed increased cardiac fibrosis in the CryAB
R120G
xSigmar1
-/-
mice hearts. Sigmar1 ablation in CryAB
R120G
mice hearts resulted in increased protein aggregate content measurement by immunostaining and aggregate size measured by electron microscopy. Sigmar1 siRNA-knockdown in CryAB
R120G
expressing cardiomyocytes also led to a dramatic increase in aggregate size and content.

Conclusion:
Overall, we found that Sigmar1 plays a vital role in the degradation of protein aggregates in the heart. Sigmar1 knockdown in CryAB
R120G
hearts promoted interstitial fibrosis, aggravated ventricular dysfunction, cardiac hypertrophy, and increased protein aggregates.
Abstract P2041: Cytochrome P450 Reductase Prevents Cardiac Hypertrophy Through Crosstalk
Authors: Lopez Melina , Malacarne Pedro F , Brandes Ralf P , Rezende Flávia
Published: November 2022

The cytochrome P450 reductase (POR) transfers electrons and thus drives the function of all cytochrome P450-dependent enzymes (CYP450). In the vascular system, the CYP450-POR system has been linked to vascular epoxyeicosatrienoic acids (EET) production and endothelial function. The functional importance of the endothelial CYP450 system in the heart, however, is unclear and was studied here, using a tissue-specific deletion approach. RNAseq revealed that murine cardiac endothelial cells express POR as well as numerous CYPs, which have been linked to different functions: CYP2 family members produce EETs, whereas CYP4 family generated omega fatty acids. Endothelial-specific, tamoxifen-inducible POR knockout mice (ecPOR
-/-
) show endothelial dysfunction and increased hypertensive response to angiotensin II infusion. ecPOR
-/-
mice also exhibited cardiac hypertrophy, an increase in cardiomyocyte area, a reduced ejection fraction and an increased left ventricle size during systole (echocardiography) as compared to control animals. RNAseq of isolated cardiac endothelial cells revealed that deletion of POR was associated with a significant decrease in genes linked to unfolded protein response (Xbp1, Hspa5) whereas genes related to cardiac development were upregulated (Sox4, Gata4). Furthermore, cardiac myocytes showed a reduced expression of genes linked to mitochondrial ATP production (Sdhaf4, Mtx1, Coq10b).Collectively, our data suggest that loss of endothelial POR promotes cardiac hypertrophy potentially by modulating paracrine signals of endothelial cells to cardiac myocytes.
Abstract P2042: Down-regulation Of Microrna-16 Improved Selenium Deficiency-induced Cardiac Dysfunction In Rats Through Wnt/β-catenin Signaling
Authors: Xing Yujie , Liu Zhongwei , Zhu Ling , Wang Xiqiang
Published: November 2022

Objective:
To investigate the mechanisms and effects of down-regulation of microRNA-16 in selenium deficiency-induced cardiac dysfunction in rats.

Methods:
Selenium deficiency was induced by low selenium diet. Injection of AAV9-carried microRNA-16 interference was used to down-regulate microRNA-16. Cardiac functions were evaluated by echocardiography and serum BNP concentrations. The expression of microRNA-16 was examined by RT-PCR. Expression levels of wnt, β-catenin, c-myc and caspase-3 were evaluated by Western blotting.

Results:
Down-regulation of microRNA-16 in rats with selenium deficiency significantly improved cardiac functions. Meanwhile, the down-regulation of microRNA-16 reduced the expression of wnt1, β-catenin and TCF in the Wnt/β-catenin signaling pathway, and the expression of c-myc and caspase-3 were also decreased.

Conclusions:
The down-regulation of microRNA-16 can improve the cardiac function of rats with selenium deficiency via the inactivation of Wnt/β-catenin signaling pathway.
Abstract P2051: Incomplete Regression And Persistence Of Interstitial Fibrosis Following Removal Of Left Ventricular Pressure Overload
Authors: Neff Lily S , Van Laer An O , Baicu Catalin F , Zile Michael R , Bradshaw Amy
Published: November 2022

Background:
Left ventricular pressure overload (LVPO) can develop from antecedent conditions, like aortic stenosis, and is associated with increased myocardial collagen content that can lead to increases in myocardial stiffness. Alleviation of LVPO in patients undergoing surgical aortic valve replacement (SAVR) resulted in reduced but persistent fibrosis evident in biopsies taken 5-yrs following SAVR. Little is known regarding cellular mechanisms that contribute to ECM turnover following hemodynamic unloading.

Objective:
Determine the time course of ECM remodeling after alleviation of LVPO.

Methods:
Transverse aortic constriction (TAC) was used to induce LVPO. Following 4wks of TAC, alleviation of LVPO was performed by removing the TAC band (unTAC). Five time points were assessed: Control no TAC, 4wk TAC, 4wk TAC+2wk unTAC, 4wk TAC+4wk unTAC, and 4wk TAC+6wk unTAC for cardiomyocyte cross-sectional area (CSA), collagen volume fraction (CVF), collagen degradation, and myocardial stiffness.

Results:
CSA increased by 47% at 4wk TAC vs control. Compared to 4wk TAC, CSA decreased by 20% at 2wk unTAC, but returned to control levels by 6wk unTAC. CVF increased by 204% at 4wk TAC vs control. Although CVF was similar between 4wk TAC and 2wk unTAC, 2wk unTAC demonstrated robust increases in markers of collagen degradation (cathepsin K, pro-matrix metalloproteinase 2 (pro-MMP2), MMP3, and MMP8). CVF significantly decreased at 4wk unTAC compared to 2wk unTAC. However, the markers of collagen degradation were reduced by 4wk- and 6wk unTAC likely contributing to persistent fibrosis. Furthermore, passive stiffness remained elevated at 6wk unTAC.

Conclusion:
In LVPO hearts, hypertrophy and fibrosis were present. Although cardiomyocyte hypertrophy fully regressed with normalization of hemodynamic load, increased myocardial stiffness and elevated interstitial fibrosis were persistent.
Abstract P2054: Epigenetic Regulation Of TLR2 By Gut Microbial Antigens Aggravates Myocardial Fibrosis Through Activating Myd88/IRAK4/NF-kB Signaling In Type 2 Diabetes
Authors: Prasad Ram , Ranjan Prabhat , Floyd Jason L , Harbour Angela , Rohella Aayush , Verma Suresh K , Grant Maria
Published: November 2022

Background:
Type 2 diabetic (T2D) patients are 40% more likely to develop cardiovascular diseases compared to healthy individuals. Previously, we have shown that circulating gut peptides such as peptidoglycan (PGN) aggravate myocardial fibrosis through DNA hypomethylation-induced activation of TLR2; however, the underlying molecular mechanisms responsible remain elusive.

Hypothesis:
Gut microbial antigens activate MyD88/IRAK4/NF-κB signaling through TLR2 in cardiac tissue, resulting in myocardial fibrosis and cardiac dysfunction in T2D.

Methods:
Gut barrier integrity, cardiac inflammation, and DNA hypomethylation (5hmC) were measured in cardiac tissue via IHC, qPCR, ELISA, and western blot analysis in
db/db
mice, a murine model of T2D with 4 months of diabetes and age-matched controls. PGN-treated cardiac fibroblasts were analyzed via in-vitro studies with and without IRAK4 and NF-κB inhibitors.

Results:
db/db mice exhibited cardiac dysfunction and myocardial fibrosis as measured by reduced LVEF and LVFS with increased LV mass and collagen synthesis. These mice also exhibited higher gut permeability as observed by reduced tight junctional proteins (ZO-1, p120-catenin, and VE-cadherin, YAP, PV1) and increased levels of plasma FABP-2. The levels of PGN were higher in plasma (p<0.003) and cardiac tissue (p<0.001) of
db/db
mice. The expression of TLR2 (p<0.003) and pro-inflammatory cytokines IL-1β (p<0.008) and TNF-α (p<0.0004) were significantly up-regulated in diabetic hearts compared to WT. Expression of 5hmC was significantly higher (p<0.001) in the hearts of diabetic mice. Increased expression of 5hmC, ten-eleven translocation 1-3 (TET-1, 2, 3), IL-1β, IL-6, and TNF-α were also observed in PGN-treated cardiac fibroblasts. IRAK4 (PF-06650883; 10μM) and NF-κB (CAPE; 50 μM) inhibition reduced the PGN-mediated fibroblast activation, resulting in reduced inflammation.

Conclusions:
Increased circulating gut microbial antigens by induction of systemic and cardiac inflammatory pathways result in damage to the myocardium of db/db mice. Therapies designed to restore the gut barrier and reduce TLR2 activation may potentially minimize the risk of diabetes-induced cardiovascular complications.
Abstract P2055: Secondary Structure And Antisense Oligonucleotides Regulate Cardiac Messenger RNA Translation And Modulates Cardiomyocyte Hypertrophy
Authors: Yao Peng , Hedaya Omar , VENKATA KADIAM , jiang feng , Wu Jiangbin , Khor EngSoon
Published: November 2022

Translation of upstream open reading frames (uORFs) typically abrogates translation for the main ORF (mORF) and regulates protein expression. uORFs are present in nearly 50% of messenger RNA (mRNA) transcripts, but their impact and regulation on biological processes are understudied. Mining of ribosome profiling data from next-generation sequencing analysis of human and mouse hearts reveals that most translated uORFs reside within mRNAs of transcription factors, which play essential roles in cardiomyocyte (CM) development and growth. We show that the uORF start codon synergizes with an immediate downstream double-stranded RNA (dsRNA) element to activate uORF and inhibit mORF translation. Biochemical and genetic evidence support that this mechanism is utilized by mRNAs encoding multiple transcription factors such as GATA4 (GATA binding protein 4), encoding a master transcription regulator of CM differentiation and pathological hypertrophy. Intriguingly, a trans-acting regulatory factor, DEAD-box RNA helicase DDX3X, is involved in unwinding the dsRNA structure and promoting mORF translation. We develop two types of antisense oligonucleotides (ASOs) that mimic the unwinding or stabilizing of the dsRNA secondary structure, thereby suppressing or enhancing the translation of this uORF, respectively. Genetic or chemical inactivation of this GATA4 uORF using CRISPR-Cas9 or ASOs in human embryonic stem cells lead to enhanced differentiation into CMs and cellular hypertrophy. At the organismal level, short-term treatment of isoproterenol and surgery-induced cardiac hypertrophy mouse models with uORF-enhancing ASOs reduces
GATA4
mRNA translation and antagonizes cardiac hypertrophy and remodeling. As a summary, this uORF-dsRNA element is a new tunable regulator of cardiac transcription factor mRNA translation and can be targeted to alter cellular and organismal phenotypes. Moreover, mechanism-based translation-manipulating ASOs can serve as promising biotechnological tools to regulate gene expression
in vitro
and
in vivo
and can be adapted to create novel RNA-based therapeutics.
Abstract P2068: The Nuclear Transcription Factor Y Controls Cell-cycle And Metabolic Gene Transcription During Cardiac Development
Authors: Cui Miao , Liu Ning , Bassel-Duby Rhonda S , Olson Eric N
Published: November 2022

The Nuclear Transcription Factor Y (NF-Y) is a trimeric complex composed of subunits NF-Ya, NF-Yb, and NF-Yc. While all three subunits are expressed across tissues and cell types, the activity of the NF-Y complex is largely dependent on the expression of its DNA-binding subunit NF-Ya. NF-Y functions as a pioneer factor in regulating gene transcription, but its cardiac-specific role is poorly understood. Using single-nuclear RNA sequencing, we previously showed that NF-Ya is expressed in regenerative cardiomyocytes in neonatal mouse hearts and its expression is downregulated after birth. Given its high expression in embryonic hearts, we hypothesized that it plays a role during cardiac development. We discovered that mice with cardiomyocyte-specific deletion of NF-Ya using alpha-MHC-Cre die before birth. At embryonic day 15, the knockout hearts showed myocardial hypoplasia and noncompaction with decreased cardiomyocyte proliferation. Interestingly, the expression of genes regulating oxidative and glycolytic metabolism as well as genes encoding fetal contractile protein isoforms were downregulated in the knockout hearts. Using chromatin immunoprecipitation and reporter assays, we found that NF-Ya directly regulates the transcription of cell-cycle and metabolic genes while indirectly controlling fetal gene expression. Combined single-nuclear RNA and ATAC sequencing analyses revealed a precocious maturation trajectory in cardiomyocytes from NF-Ya knockout embryos, leading to a reduced reservoir of immature regenerative cardiomyocytes in the fetal heart. Our study reveals cellular heterogeneity of fetal cardiomyocytes and the role of NF-Y in regulating cardiac growth and maturation before birth. Our study also identified a previously unrecognized transcriptional control mechanism of metabolic genes controlled by the NF-Y complex and highlights the importance of both oxidative and glycolytic metabolism during fetal heart development and maturation.
Abstract P2069: In Vivo Investigation Of The Mechanisms Regulating Truncated Dystrophin Protein Turnover In The Heart
Authors: Bez Batti Angulski Spies Addeli , Bauer John , Metzger Joseph M
Published: November 2022

Numerous inherited and acquired cardiac diseases are directly linked to a deficiency in the protein dystrophin, such as the development of severe cardiomyopathy in Duchenne Muscular Dystrophy (DMD) patients. Dilated cardiomyopathy, arrhythmias, and congestive heart failure represent the most important life-limiting condition in DMD. Currently, there is great excitement in the muscular dystrophy community as multiple ongoing gene-based therapies, are advancing through clinical trials. These therapies utilize miniaturized dystrophin constructs, and these modified dystrophins are the focus of this study. We implemented an innovative and valuable system to determine the direct physiological significance of dystrophin protein deficiency including determining the half-life of truncated dystrophin constructs. This system provides a novel means to investigate mechanistically how deletions in dystrophin affect therapeutically shortened dystrophin turnover in cardiac muscle in vivo. We used a floxed allele approach together with a cardiac directed (αMHC Mer-Cre-Mer) inducible Cre for precise control of full-length dystrophin or therapeutic micro-dystrophin gene excision. We examined the time course of full-length and micro-dystrophin mRNA as a biologically relevant surrogate for intact gene excision efficiency. Our data showed evidence of significant full-length dystrophin cDNA and micro-dystrophin gene excision, with a complete loss of micro-dystrophin mRNA and a gene excision efficiency of 80% for full-length dystrophin. Heart tissues were then extracted for protein quantitative analysis of micro-dystrophin (5, 10 and 30 days) and of full-length dystrophin (1, 3 and 6 months) post tamoxifen administration. Results reported a fast turnover rate for micro-dystrophin in the heart, with calculated half-life of between 5-7 days. In marked contrast, full-length dystrophin was highly stable with dystrophin protein content (~ 40%) at 6 months, after dystrophin gene excision. Studies of in vivo full-length and micro-dystrophin protein half-life in skeletal muscle, are ongoing. This work will provide key information required for long-term success of ongoing and future DMD therapies featuring gene therapy with shortened dystrophins.
Abstract P2070: Insights Into RBM20 Cardiomyopathy Revealed By Global Proteomics
Authors: Gregorich Zachery R , Larson Eli J , Zhang Yanghai , Ge Ying , Guo Wei
Published: November 2022

Patients with missense mutations in RNA binding motif protein 20 (RBM20), an RNA binding protein and splicing factor, develop a clinically aggressive form of dilated cardiomyopathy (DCM). Recent studies revealed that DCM-linked RBM20 missense mutations promote re-localization of the protein from the nucleus to the cytoplasm where it forms ribonucleoprotein (RNP) granules—non-membrane bound assemblies formed from RNAs and RNA regulatory proteins. The sequestration of RNAs and RNA regulatory proteins in these granules has the potential to dramatically alter the landscape of protein expression in the cardiomyocyte; yet, how RBM20 missense mutations impact global gene expression at the protein level has not been explored. Herein, we utilized mass spectrometry (MS)-based proteomics to identify global changes in protein expression in cardiac tissue harvested from mutation knock-in (KI) mice harboring the DCM-linked S637A and S639G RBM20 mutations. Our analysis revealed a total of 212 and 190 differentially expressed proteins (DEPs) in S637A and S639G KI mice, respectively, relative to wild-type. In general, identified DEPs were similar between KI mice with 81 and 38 proteins commonly up- and down-regulated, respectively. Consistent with mutant RBM20-mediated granule formation, 15 and 12 of the up-regulated proteins in S637A and S639G KI mice, respectively, have previously been implicated in granule assembly. Other up-regulated proteins in KI mice included chaperones and cytoskeletal proteins, as well as proteins involved in RNA metabolism and metabolite interconversion. Interestingly, proteins involved in the regulation of metabolite interconversion were also the most common protein class down-regulated in both KI mice, suggesting that broad changes in cellular metabolism may be a common feature associated with the RBM20 S637A and S639G missense mutations. Additionally, our proteomics analysis confirmed the differential expression of approximately 20-30% of previously identified differentially expressed genes based on RNAseq analyses from S637A and S639G KI mice. Our results provide the first glimpse of global changes in gene expression at the protein level associated with the RBM20 S637A and S639G missense mutations.
Abstract P2077: Caspase8ap2 M6a Mrna Methylation Enhances Cardiomyocyte Apoptosis Following Myocardial Ischemia
Authors: Dutta Roshan Kumar K , Ranjan Prabhat , zhang qinkun , Krishnamurthy Prasanna , Lal Hind , Young Martin E , Verma Suresh
Published: November 2022

Objective:
Heart failure is the leading cause of morbidity and mortality in the United States. Cardiomyocyte death is one of the critical mediators of cardiac dysfunction following ischemic injury. However, the role of posttranscriptional regulation on cell-death-associated genes is not well explored during ischemic heart failure. N6-methyladenosine (m6A) is the most abundant and conserved chemical modification found in eukaryotic mRNA and is associated with mRNA synthesis and metabolism. We have recently shown that m6A RNA methylation regulates the angiogenic potential of endothelial cells following ischemic injury. However, the role of m6A mRNA methylation in cardiac cell death following ischemic injury is not well defined.

Methods:
Cardiac function (using echocardiography) was measured at baseline and four weeks post-MI in mice. In the end, mice were euthanized, and hearts were excised for biochemical and histological analysis. Cardiac cell death was assessed in H9C2 cells and cardiomyocytes using FACs (Annexin V and PI) and TUNNEL assay. The Casp8AP2 stability were measured using actinomycin D assay. To demonstrate the molecular mechanism, METTL3 was inhibited using siRNA or shRNA.

Results:
The cardiac function was significantly reduced following MI. Enhanced m6A mRNA methylation was observed in mice hearts following MI. Histological (TUNEL assay) and biochemical (WB: Caspase 8, cleaved cas8, caspase3, cleaved caspase3, Casp8AP2) analysis showed increased myocytes apoptosis in mice following MI. At the molecular level, hypoxic stress (nutrient depiviation+1%O2) induces global m6A mRNA methylation, which leads to Cas8AP2 gene stability and activation of extrinsic apoptotic pathways in cardiomyocytes in vitro. In contrast, METTL3 inhibition significantly reduced Cas8AP2 mRNA methylation, enhanced its degradation, and ultimately reduced cardiomyocyte cell death.

Conclusion:
In conclusion, we have shown that increased Casp8AP2 m6A RNA methylation enhances its stability and activates caspase-3 cleavage, which eventually leads to cardiomyocyte death following myocardial ischemia. The reduction of the RNA methylation through METTL3 inhibition (methyltransferase) reduced hypoxia-induced apoptosis of cardiomyocytes.
Abstract P2081: Beclin1 Ameliorates The HIV-1 Induced Cardiac Dysfunction In Mice Model Of HIV
Authors: Kashyap Shiridhar , Rabbani Maryam , Futuro de Lima Isabela Beatriz , Gawasker Sameep , Patel Raj , Rappaport Jay , Liang Qiangrong , Gupta Manish K
Published: November 2022

Background:
Despite antiretroviral drugs-mediated control of active HIV infection, cardiovascular disease (CVD) remains one of the leading causes of comorbidity in people living with HIV/AIDS (PLWHA). Recent studies suggest that activation of the immune system and chronic inflammation increases CVD risk in PLWHA. Dysregulation of the immune system can interact with several cellular functions, including autophagy and mitophagy. The autophagic factor Beclin1 is a crucial regulator of cellular autophagy and mitophagy. In this study, we tested the role of Beclin1 in cardiac function using a mice model of HIV.

Methods and Results:
This study was performed using transgenic mice of HIV-1 Tg26 mice, which was constructed before using HIV-1 DNA with deletion of 3-kb region of the gag/pol genes. Tg26 mice were crossed with Beclin1 transgenic mice to test the role of Beclin1. Cardiac function of Tg26 and Tg26 mice crossed with Beclin1 was evaluated by echocardiography. Cardiac autophagy was determined by western blots using autophagy regulatory markers. Our data suggest that Tg26 mice have significantly reduced cardiac function. Further, Tg26 mice show increased reactive Oxygen species (ROS) level, accumulation of LC3II, increased P62 level, and reduced autophagy flux. Additionally, western blots with cardiac tissue show upregulation of electron transport chain (ETC) complex proteins of mitochondria in the Tg26 mice that may be due to defective mitochondria accumulation in Tg26 mice heart. Interestingly, Beclin1 expression can improve the cardiac function of Tg26 mice heart. Mechanistically, we found that Beclin1 overexpression can restore the autophagy flux and reduce the cellular ROS level in the heart of Tg26 mice.

Conclusion:
Our study suggests that Beclin1 plays critical role in regulation of cardiac function during HIV induced cardiomyopathy and can be used as a potential therapeutic tool to control comorbidity in the PLWHA.