bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 457: Protein Arginine Methyltransferase-1 is Required for Epicardial Epithelial-to-Mesenchymal Transition
Authors: Jackson-Weaver Olan , Wu Jian , Gou Yongchao , Shi Shihong , Sucov Henry , Xu Jian
Published: June 2021

Rationale:
Epicardial epithelial-to-mesenchymal trasition (EMT) is a vital process in embryonic heart development. During EMT, epicardial cells acquire migratory and invasive properties, and differentiate into new cell types, including cardiac fibroblasts and coronary smooth muscle cells. EMT is characterized by an increase in mesenchymal proteins such as Slug and Fibronectin, and a decrease in cell-junction proteins such as E-Cadherin, and is dependent on TGF-β signaling. We have recently demonstrated that protein arginine methyltransferase-1 (PRMT1) is necessary for TGF-β family signaling and EMT in non-epicardial cell types.

Objective:
To determine the role of PRMT1 in epicardial EMT.

Methods and Results:
We investigated the role of PRMT1 in epicardial EMT in mouse epicardial cells. PRMT1 siRNA prevented the increase in Slug and Fibronectin and the decrease in E-Cadherin in TGF-β treatment-induced EMT of mouse epicardial cell line MEC1. PRMT1 siRNA also reduced the migration and invasion of MEC1 cells. These results demonstrate that PRMT1 is required for epicardial EMT. In WT1-Cre
ERT
;ROSA-YFP
fl/fl
mouse embryos, PRMT1 siRNA reduced epicardial EMT in a thorax culture model. Among the key transcription factors that regulate the EMT program, Slug, but not Snail, is specifically regulated by PRMT1. We further identified that PRMT1 siRNA also increased the expression of p53, a key regulator of the Slug degradation pathway. PRMT1 siRNA increases p53 expression by decreasing p53 degradation, and shifted p53 localization to the cytoplasm.
In vitro
methylation assays further demonstrated that PRMT1 methylates p53. Knockdown of p53 increased Slug levels and enhanced EMT, establishing p53 as a regulator of epicardial EMT through controlling Slug expression.

Conclusions:
The PRMT1-p53-Slug pathway is necessary for epicardial EMT in cultured MEC1 cells as well as in the epicardium
ex vivo
.
Abstract 458: Novel Interactions With AMP-activated Protein Kinase Identified by Promiscuous Biotin Ligase Assay
Authors: Lipovka Yulia , Konhilas John P
Published: June 2021

Estrogen is positioned to play a unique role in cardiovascular disease (CVD) since it can respond to environmental, genetic and non-genetic cues to impact gene expression and cellular signaling. Recently AMP-activated protein kinase (AMPK) has been identified as an important modulator of CVD. We propose that AMPK represents a nodal point for estrogen-dependent modulation of CVD. Our preliminary studies in a non-muscle cell line demonstrate that estrogen (E2) regulates AMPK activity predominantly through Estrogen Receptor alpha (ERα). In addition, both classical estrogen receptors (ERα and ERβ) bind to the α-catalytic subunit of AMPK, and the upstream kinase complex LKB1 is required for AMPK activation after E2 stimulation.

The next step is to translate these findings into the cardiac system. In order to do that, we validated a novel technology to screen for interactions based on a promiscuous biotin ligase from
E. coli
. We created vectors containing our protein of interest, AMPK and/or the upstream kinase complex, LKB1, fused to the biotin ligase. After infecting Neonatal Rat Cardiomyocytes (NRCM) with viruses expressing those constructs, we added biotin to the cells, allowing biotinylation of proximal endogenous proteins; following biotin-affinity capture and western blot analysis. We show that estrogen receptors bind both to the α-catalytic subunit of AMPK, and the upstream kinase complex LKB1 in NRCM. Co-immunoprecipitation of endogenous proteins confirmed those interactions. In addition, we characterized a novel interaction between AMPK and the cytoskeletal protein, vinculin. Previous studies in our lab showed that AMPK is a modulator of myofilament function, and that the estrogen receptors associate with myofilament proteins. These, along with the findings detailed above open a new perspective on cardiac energy regulation.
Abstract 461: Microrna-126 Overexpression Rescues Impaired Efferocytosis in Diabetes
Authors: Suresh Babu Sahana , Thandavarayan Rajarajan A , Joladarashi Darukeshwara , Jeyabal Prince , Krishnamurthy Shashirekha , Bhimaraj Arvind , Youker Keith A , Krishnamurthy* Prasanna
Published: June 2021

Cardiac healing after myocardial ischemia requires accumulation of macrophages at the site of injury for the clearance of apoptotic/dead cardiomyocytes, a process known as efferocytosis. Effective clearance of apoptotic cells in turn results in inflammation resolution during cardiac regeneration. Diabetes accompanied by hyperglycemia is known to compromise the function of macrophages leading to impaired inflammation resolution during cardiac regeneration, wound healing, atherosclerosis and auto-immune disorders. However, the effect of diabetes on macrophage efferocytosis and molecular mechanisms involved have not been understood so far. We found miR-126 is essential for the macrophage efferocytosis of apoptotic myocytes in high glucose conditions (HG). In-vivo, miR-126 expression was reduced and, a corresponding increase in A Disintegrin And Metalloprotease 9 (ADAM9) expression was observed in human diabetic heart failure tissues compared with the normal counterparts. In-vitro, macrophages efferocytosis was reduced in HG compared to normal glucose. Over-expression of miR-126 increased the efferocytosis in HG. Mechanistically, reduced miR-126 leads to increased expression of human ADAM9. ADAM9 in turn cleaves Mer tyrosine receptor kinase (MerTK), a proto-oncogene that plays a role in efferocytosis, leading to the formation of soluble-Mer that reduces the macrophage efferocytosis in HG conditions. This, we term as
“not ready to eat”
signal of the macrophages, which could be rescued by overexpressing miR-126 and termed as
“ready to eat”
signal of the macrophages.
Abstract 462: Activation of Akt2 by Apolipoprotein E Protects ATP-binding Cassette A1 From Degradation
Authors: Yang Hong , Okoro Emmanuel U , Guo Zhongmao
Published: June 2021

We previously reported that lipid-free apolipoprotein E (apoE) is able to increase ATP-binding cassette transporter A1 (ABCA1) transcription by activation of a signaling cascade involving very low-density lipoprotein receptor, apoE receptor 2, disabled-1, phosphatidylinositol 3-kinase (PI3K), protein kinase Cζ, and specificity protein 1 (Sp1). Here we demonstrated that treatment of RAW 264.7 murine macrophages with human apoE3 enhanced Akt phosphorylation, and upregulated ABCA1 protein and mRNA expression. Inhibition of PI3K weakened apoE3-induced Akt phosphorylation, and reduced ABCA1 protein and mRNA levels. In contrast, inhibition of Akt only diminished apoE-induced ABCA1 protein but not significantly alter ABCA1 mRNA level. Inhibition of protein synthesis did not erase the ability of apoE3 to increase ABCA1 protein level. Further, apoE3 increased the resistance of ABCA1 protein to calpain-mediated degradation without affecting calpain activity. Treatment of 264.7 cells with apoE3 selectively enhanced the phosphorylation of Akt1 and Akt2 but not Akt3. Knockdown of Akt1 enhanced the phosphorylation of Akt2. Vice versa, knockdown of Akt2 enhanced Akt1 phosphorylation. Underexpression of Akt1 or Akt2, respectively, increased and decreased ABCA1 protein level; while overexpression of these Akt isoenzymes caused changes in ABCA1 protein level opposite to those induced by knockdown of the corresponding Akt. These data imply that apoE3 guards against calpain-mediated ABCA1 degradation through Akt2. PI3K might be a step in the signaling pathway for apoE3 to trigger Akt2. [This study was supported by NIH grants SC1HL101431].
Abstract 463: Elevated Oxidative Stress and Reduction of Hydrogen Sulfide (H
2
S) and Nitric Oxide (NO) in HIV Patients: Possible Role in Cardiovascular (CV) Disease
Authors: Islam Kazi N , Donnelly Erinn , Parsons Christopher , Lefer David J
Published: June 2021

Background:
HIV attacks the immune system and weakens the ability to fight infections/disease. HIV infection confers approximately two-fold higher risk of cardiac events compared with the general population. ADMA, high-sensitivity CRP (hs-CRP) and IL-6 are well-characterized biomarkers of cardiac events. H
2
S and NO exert potent antioxidant properties and are cardioprotective. The present study was undertaken to determine the levels of H
2
S, NO, and CV disease biomarkers in HIV patients.

Methods:
Plasma from both HIV patients and normal subjects were used for all assays. Oxidative stress was determined by analyzing the levels of advanced oxidation protein products and H
2
O
2
. Antioxidant levels were determined by measuring the levels of trolox equivalent capacity. ADMA, hs-CRP, and IL-6 were determined by using ELISA. The levels of H
2
S/sulfane sulfur and nitrite were determined in plasma by using gas chromatography and HPLC, respectively.

Results:
We observed a marked induction in the levels of oxidative stress and decreased antioxidant status in the plasma of HIV patients as compared with the controls. CV risk factors levels such as ADMA, hs-CRP, and IL-6 were significantly increased in the plasma of HIV patients. Significant reduction in the levels of both nitrite and H
2
S/sulfane sulfur were observed in the plasma of HIV patients as compared with controls.

Conclusion:
Our data demonstrate significant increases in oxidative stress and CV disease biomarkers in conjunction with decreased bioavailability of H
2
S and NO in HIV patients. Diminished levels of these two cardioprotective gaseous signaling molecules may be involved in the pathogenesis of CV disease in the setting of HIV.
Abstract 467: Glucose Promotes M2-related Transcriptional Reprogramming of Resident Cardiac Macrophages in Response to Hypothermic Ischemic Arrest and Reperfusion
Authors: Bux Ahmed , Vasquez Hernan , Taegtmeyer Heinrich , Harmancey Romain
Published: June 2021

Stress hyperglycemia and inflammation frequently develop in open heart surgery patients. Although both factors independently contribute to increased peri-operative morbidity and mortality, the impact of high glucose levels on cardiac inflammatory response remains unknown. We investigated the isolated working rat heart as a model to study cardiac early stress response to surgery. Hearts of male Sprague Dawley rats were cold-arrested and subjected to 60 minutes normothermic reperfusion in the working mode with Krebs-Henseleit buffer supplemented with ketone bodies and propionate plus glucose (25 mM) or mannitol (25 mM; osmotic control). Alterations of gene expression in the left ventricle were determined by microarray and real-time PCR analyses. Compared to non-perfused hearts, perfused hearts displayed a more than twofold increased expression for 71 genes (mannitol group) and 103 genes (glucose group) connected to inflammation, cell proliferation, and apoptosis. The transcriptional changes were highly similar to gene alterations previously reported in the right atrium (
P
< 2.34E-16) and left ventricle (
P
< 4.83E-46) of patients who underwent cardiac surgery with cardiopulmonary bypass. Pathway analysis with Reactome revealed an up-regulation of metabolic processes associated with the proliferation and activation of immune cells, including glycolysis, glutaminolysis, fatty acid synthesis, polyamine synthesis, and hexosamine synthesis. Although the transcriptional remodeling occurred independently from the presence of glucose, glucose significantly increased further the expression of several transcription factors and markers associated with M2 polarization of macrophages, including Myc (1.6-fold), Nr4a1 (1.3-fold), Nr4a2 (1.8-fold), Zc3h12a (1.3-fold), Fosl2 (1.4-fold), Cebpb (1.2-fold), and Arg1 (1.7-fold). Interestingly, glucose failed to enhance the expression of M2-related genes in the heart of rats rendered insulin resistant by high-sucrose feeding. Besides demonstrating that the isolated working rat heart accurately reproduces the stress response associated with open heart surgery, the results also suggest that glucose promotes the alternative activation of resident cardiac macrophages in the stressed heart.
Abstract 47: Treatment With a DNA Vaccine in Experimentally
Trypanosoma
cruzi
-infected Dogs Modulates Cellular Immune Response
Authors: Arce-Fonseca Minerva , Carbajal-Hernández Ana C , Lozano-Camacho Mónica , Carrillo-Sánchez Silvia C , Rios-Castro Martha , Rodríguez-Morales Olivia
Published: June 2021

Chagas Disease (CD) is caused by the protozoan
Trypanosoma
cruzi
. Eight million people are infected in Latin America and 10,000 die annually; because CD has prolonged chronicity is considered the parasitic disease with greater economic burden in Latin America.

The 30% to 40% of patients develop chronic chagasic cardiomyopathies and disorders of the gastrointestinal tract.

It has not found an effective treatment to cure the chronic disease; nifurtimox and benznidazole in the acute stage offer controversial results.

The objective of this study was to test a treatment with plasmid DNA containing
T.
cruzi
genes in dogs with experimental CD and to determine the cellular immune response.

Thirty Beagle dogs were divided into 6 groups with 5 dogs each. Dogs from five groups were intraperitoneally infected with 3500 metacyclic trypomastigotes / kg body weight. Two weeks after infection, intramuscular therapeutic vaccinations were administered thrice at 2-week intervals. The plasmids used were: pBCSSP4 (containing a gene encoding an amastigote-specific protein), pBCSP (containing a gene encoding a
trans
-sialidase protein), pBCSSP4 + pBCSP and pBK-CMV (empty vector); control groups were saline solution
mock-
treated and non-infected/non-treated dogs. IL-1, IL-6, IL-12, IFN-gamma and TNF-alpha levels were determined by ELISA in each of the serum samples at different times. The proliferative response of spleen cells
in vitro
at 15 and 30 days after last treatment was studied.

IL-6 and IL-12 levels were slightly increased in pBCSSP4 plasmid-treated animals; TNF-alpha and IFN-gamma levels rose after pBCSP plasmid treatment. Treatment with either two recombinant plasmids produced no increase in IL-1 levels. The use of mixed plasmid did not have a synergistic effect.

Cell proliferation was induced by DNA plasmid treatment. The highest stimulation index (2.5) was observed in pBCSSP4 plasmid-treated dogs.

In conclusion, cellular immune responses are stimulated by therapeutic DNA vaccine; pBCSP plasmid treatment showed polarized type Th1 immune response, while there was a balance in Th1 / Th2 response when pBCSSP4 plasmid was used as treatment.

These results support the promising novel therapeutic application with DNA using
TcSSP4
and
TcSP
genes against CD.
Abstract 48: Differentiation of Moderate and Severe Ischemic Heart Failure by Invasive and Non-invasive Assessments of Diastolic Function in a Rat Model of Chronic Heart Failure
Authors: Mohran Saffie , Lancaster Jordan , Sanchez Pablo , Goldman Steven , Juneman Elizabeth
Published: June 2021

Background:
This work is designed to determine if specific left ventricle (LV) pressure-volume relations, hemodynamic, and echo derived parameters of diastolic function are able to separate severe from moderate CHF in rats with left coronary artery occlusion.

Hypothesis:
Echocardiographic indices of diastolic function, end-diastolic pressure (EDP), dead volume, stiffness constants (k), and pressure volume relations predict the severity of CHF in infarcted rats.

Methods:
Male Sprague Dawley rats (N=14) were randomized to undergo left coronary artery ligation or sham operation. Echocardiography was performed at 3 and 6 weeks post coronary ligation. The rats were categorized into moderate or severe CHF according to their LVEDP at 6 weeks post ligation. Invasive hemodynamic measurements with solid state micro manometer pressure catheters as well as diastolic pressure-volume relation values were obtained at the 6 week end point.

Results:
Moderate and severe CHF rats had significantly (P<0.05) elevated left ventricular (LV) end-diastolic pressure (LV EDPs), prolonged time constants of LV relaxation (tau), and decreased peak development pressures. When moderate versus severe CHF rats were separated based on LV EDP, early diastolic anterior wall radial relaxation velocity as well as e’, and E/e’ had strong correlations with invasive hemodynamic measurements of diastolic functions. There was a trend towards decreased compliance as measured by stiffness constants in severe heart failure group. Differences (P<0.05) in dead volume, mean arterial pressure (MAP), tau, and ejection fraction (EF) were also displayed. End diastolic pressure-volume analyses illustrated significant differences in plot positioning and curvature.

Conclusion:
While it is possible to separate rats with moderate and severe CHF in the rat coronary artery ligation model, the separation is not simply based on a specific EF value. This work may be useful in deciding whether there is a differential effect of new treatments for severe versus moderate CHF.
Abstract 52: Isolation, Characterization and Partial Direct Differentiation of Patient Derived Human Primary Cardiac and Placental Fibroblasts From Surgical Discard Tissue into Induced Mesoderm Progenitor Cell
Authors: Sadeghi Pour Pouya , Emanueli Costanza , Caputo Massimo , Angelini Gianni
Published: June 2021

Background:
Human primary cells more closely mimic the physiological state of cells in vivo and generate more relevant data representing living systems. Access to the surgical discard tissue enables the possibility of patient derived, disease specific, cellular bio-bank. In achieving this goal, target cellular lines must be isolated, characterized and fully expanded under specific Standard Operating Procedures(SOP). In this project, we aim in achieving and implementing such goal for Fibroblasts derived from two distinct patient tissues. In parallel, in light of cardiac regenerative therapy, with reference to the published work on Cardiomyocyte Direct Differentiation on mouse fibroblasts into induced cardiomyocyte-like cells (iCMs) by transduction with MicroRNAs (1,133a, 208a and 499) we sought to determine whether primary human cardiac and placental fibroblasts could have the potential in direct differentiation to induced cardiomyocyte-like cells (iCM) through sequential administration of small molecule (ITD-1) in combination with MicroRNAs.

Methods and Results:
Primary right atrial appendage cardiac and smooth chorion laeve placental fibroblast isolates were generated from surgical discard patient derived tissues subjected for identification of the most efficient isolation method through available enzymatic and mechanical techniques. We have shown through FACS analysis that both groups extracellular marker expression can be limited to CD90+CD31-CD44+FBSP+ which constitutes the majority of cultured primary fibroblasts. Respectively, intercellular expression of DDR2+ VIM+ COL1+CD90+ markers was validated through immunofluorescence microscopy. Transcriptional profiling of Noted lines showed expression of Fibroblast specific genes at higher rate.Furthermore, sorted placental fibroblast isolates were subjected to (ITD-1)treatment and mature MicroRNA mimic cocktail. Transcriptional profiling analysis through RT-PCR concluded down regulation of most distinct fibroblast markers and partial expression of MESP1.
Abstract 53: Characterization of a Feline HFpEF Model Induced by Slow Progressive Pressure Overload
Authors: Wallner Markus , Berretta Remus M , Sun Shu-Yu , Small Kersten M , Madwed Jeffrey B , Troupes Constantine D , Sharp Thomas E , Borghetti Giulia , Oyama Mark A , Fox Philip R , Houser Steven R
Published: June 2021

Rationale:
The prevalence of Heart Failure with preserved Ejection Fraction (HFpEF) is almost equal to that of Heart Failure with reduced Ejection Fraction (HFrEF) and represents a major public health problem. There are no proven effective treatments for HFpEF, partially attributable to the lack of well-established animal models for HFpEF.

Hypothesis:
Cats develop a HFpEF phenotype similar to patients after induction of slow progressive pressure overload.

Methods and Results:
Female and male short hair cats, aged 2 months, underwent either aortic constriction, with a customized pre-shaped band, or sham procedure. In contrast to immediate pressure overload, this approach results in a slow progressive pressure overload during growth. Transthoracic echocardiography was performed at baseline and 1, 2, 3, and 4 months after banding. Concentric LV hypertrophy was observed and heart weight to body weight ratio significantly increased in banded male and female cats compared to sham (male: 8.5±1.6 vs. 4.5±0.4; female: 7.4±0.2 vs. 4.9±0.8; p<0.05). Wet/dry lung weight ratio, indicating pulmonary congestion, did not differ between groups. End-diastolic wall thickness was significantly increased in banded cats at 2, 3 and 4 months, while the end-diastolic diameter and systolic function did not differ between banded and sham cats at any time point. A significant left atrial (LA) enlargement was observed in banded cats at 3 and 4 months, as measured by an increased LA/aorta ratio and increased LA area. LA ejection fraction was significantly decreased after aortic constriction (male sham vs. banded: 75.4±0.2 vs. 47.3±0.2; p<0.01; female sham vs. band: 75.4±2.1 vs. 53.9±4.4; p<0.05). Diastolic function, assessed by tissue Doppler imaging, was impaired in banded cats.

Summary and Conclusion:
Slow progressive pressure overload in cats induces severe LV concentric hypertrophy, LA enlargement and dysfunction without causing LV dilation or systolic dysfunction, mimicking many clinical phenotypes in HFpEF patients. Further characterization will include incorporating invasive measurements, exercise capacity and serum biomarkers and testing of novel therapies.
Abstract 57: Myofibrils From Pediatric IDC Patients Display Faster Relaxation Kinetics and Reduced Tension Generation
Authors: Jeong Mark , Sucharov Kika , Stauffer Brian , Ambardekar Amrut , Lin Yin , Mahaffey Jennifer , Miyamoto Shelley
Published: June 2021

Objective:
Pediatric patients with heart failure, compared to adults, respond differently to current armamentarium of heart failure (HF) therapeutics, indicating differences in the hearts of children and adults. We examined myofibril mechanics in children and adults with and without HF to determine: 1) baseline myofibril characteristics, 2) differences between children and adults, and 3) myofibril mechanics in the setting of HF.

Methods:
Myofibrils were obtained from pediatric and adult patients with IDC undergoing cardiac transplantation. Unused donor hearts were used as non-failing control (NF). Written informed consent was obtained. Myofibrils were stretched from slack length and set 5-10% above slack sarcomere length (SL), and Ca
2+
-activated and relaxed at 15°C. Mechanical and kinetic parameters: maximal tension (T
max
, mN/mm
2
); rate constant of tension development (
k
ACT
, S
-1
); duration of slow relaxation phase (
t
LIN
, mSec), rate constant of fast phase relaxation (
k
REL
, S
-1
). Mechanical parameters ± SEM are shown.

Results:
Compared to NF adults, myofibrils from NF pediatric hearts had shorter SL (2.04±0.01 μm vs 2.11±0.01, p < 0.0001), slower
t
LIN
(202.7+9.0 msec vs 152.9+4.1, p < 0.0001) and slower
k
REL
(7.8+0.4 S
-1
vs 15.7+0.8, p < 0.0001). T
max
and
k
ACT
were similar. In the adult cohort, myofibrils from adult IDC patients showed slower
k
ACT
(0.45+0.02 S
-1
vs 0.57+0.03, p < 0.005) and slower
k
REL
(11.9+0.7 S
-1
vs 15.7+0.8, p < 0.005). T
max
and
t
LIN
were similar. In the pediatric cohort, myofibrils from IDC developed lower T
max
(35.3+2.5 mN/mm
2
vs 57.5+4.4, p < 0.0001), and faster
t
LIN
(136.6+3.2 msec vs 202.3+8.9, p < 0.0001) and faster
k
REL
(9.1+0.4 S
-1
vs 7.8+0.4, p < 0.05).
k
ACT
was similar.

Conclusion:
NF myofibrils from children have shorter resting SL and slower relaxation kinetics than NF adults. While myofibrils from adults IDC result in slower activation and exponential relaxation kinetics, children with IDC display faster relaxation kinetic and reduced tension generation. These results show that myofibrils from children have fundamental differences in mechanical characteristics at baseline and in the setting of heart failure.
Abstract 58: Differential Ventricular Remodeling Induced by Thin Filament Mutational Effects on the Tropomyosin Overlap Structure
Authors: McConnell Mark , Tal-Grinspan Lauren , Lynn Melissa , Schwartz Benjamin , Fass Ofer , Farah Hassan-Galaydh , Deranek Andrea , Jayasundar Jayant James , Tardiff Jil C
Published: June 2021

Inherited mutations in cardiac thin filament proteins create primary alterations in structure. These changes then lead to pathologic remodeling of the heart. We have proposed that two mutations, tropomyosin (Tm) D230N which leads to dilated cardiomyopathy (DCM) and cardiac troponin T (cTnT) R92L, which leads to hypertrophic cardiomyopathy (HCM), directly affect the Tm overlap, a crucial structure regulating myofilament activation. These mutations lead to divergent ventricular remodeling, which we hypothesize is caused by differential effects on the Tm overlap, an inherently dynamic system. In order to investigate this hypothesis, we used time-resolved Forster resonance energy transfer (FRET) to measure discrete distances across the proteins of the overlap, and differential scanning calorimetry (DSC) in order to characterize the baseline structure of this region as well as the effects of mutants. We found that the cTnT R92L mutant increased interprotein distance at the proximal N-terminal end of this region. In contrast, the Tm D230N mutant decreases interprotein distance at the proximal C-terminal end of the overlap. DSC studies showed a significant increase in the association of Tm with actin in the presence of Tm D230N, predicted to increase cooperativity of thin filament activation. These finding are in agreement both with the human population who carry the disease, as well as the mouse lines in our lab, which phenocopy the human disease. Overall, the study further characterized the wild type structure of the Tm overlap, and revealed the differential structural effects of the mutants, thereby, for the first time linking local changes in structure to phenotypic changes and ventricular remodeling leading to HCM and DCM. Further studies will investigate the role of isoform and phosphorylation modifiers, elucidating how these modify human disease in the presence and absence of cardiomyopathy linked mutations.
Abstract 60: Double Hit Mutations in Thoracic Aortic Aneurysm Disease That Have Variable Impact on Phenotype
Authors: Hambly Brett D , Robertson Elizabeth , Portelli Stefanie , Lu Yaxin , Kekic Murat , Jeremy Richmond
Published: June 2021

Multiple genes are associated with thoracic aortic aneurysm (TAA) formation, including FBN1, TGFBR1&2, COL3A1 & 5A2, and MYH11. Given the population incidence of mutations in these genes we hypothesise that it is likely that ‘double hit’ mutations occur, which may result in an altered clinical phenotype when compared with the single gene mutation.

Genomic DNA analysis of 19 genes implicated in TAA was performed on 12 patients with inherited TAA disease. Of the patients analysed, one patient (P1) was identified as having a mutation in both MYH11 (c.2517G.C (pW839C)) and FBN1 (splice donor site c.4210+1G>A). Another patient (P2) was identified as having both COL5A2 (c.3794A>G (pD1265G)) and FBN1 (c.5861T>G (p.F1954C)). All mutations were predicted by PolyPhen to be deleterious.

P1 has Marfan Syndrome (MFS), caused by the FBN1 mutation, and has severe aortic dilatation while his mother (M1), who also has MFS but does not have the MHY11 mutation, developed mild aortic dilatation, suggesting that the MHY11 mutation in P1 has resulted in a more aggressive phenotype.

P2, siblings S1 and S2 and father (F2) all have relatively less severe MFS with progressive moderate dilatation of the aorta. Thus, the phenotype of pedigree 2 is similar for all affected members, but only P2 has a double hit mutation, suggesting that the presence of the COL5A2 mutation in P2 has not altered the clinical phenotype.

This is the first instance where double hit mutations have been described in inherited thoracic aortic aneurysm but the effect of multiple mutations on phenotype appears to be variable. Mutation analysis for individuals with inherited thoracic aortic aneurysm should consider the possibility of multiple gene culprits.
Abstract 64: An Integrated Model for
Titin
Truncation Mutation Interpretation
Authors: Zou Jun , Tran Diana , Pelonero Angelo , Deo Rahul C
Published: June 2021

Background:
We recently discovered a conserved internal promoter in the
Titin
gene, which explains why truncating mutations in the C-terminal two thirds of the zebrafish
ttna
protein result in more severe disease, recapitulating a puzzling observation in human dilated cardiomyopathy (DCM) patients. Here we focus on the contribution of alternative splicing to the DCM phenotype, both in zebrafish
Titin
truncation mutants and in the context of an integrative model for
Titin
mutation interpretation.

Methods and Results:
Using CRISPR/Cas9, we disrupted an alternatively spliced exon in the I-band of
Titin
, normally present in zebrafish heart but absent in skeletal muscle. The resulting mutants had, on average, a milder cardiac phenotype than those with mutations in constitutive exons but also showed striking inter-sibling variability in disease expression, ranging from intact cardiac blood flow to severe early demise. The mutant exon demonstrated nonsense-altered splicing and disease severity paralleled selective deficiency in
Titin
transcript level, implying that variability in mutated exon inclusion coupled with nonsense-mediated decay (NMD) modulated phenotype. We next amassed
Titin
mutation information from 1785 human DCM cases and >68,000 controls to model mutation distribution and found three variance components 1) splicing; 2) internal isoform disruption; and 3) targeting of the C-terminal 2000 amino acids. An integrated model demonstrated strong predictive performance with an area under the receiver operating characteristic curve of 0.79 and correctly identified the highest risk individuals.

Conclusions:
We conclude that genetically targeted models and large-scale human data can be complementary in overcoming the challenges of genetic data interpretation.
Abstract 67: Metabolic Programming Regulates Proliferation and Binucleation of Postnatal Cardiomyocytes
Authors: Liccardo Daniela , LaCanna Ryan , Tian Ying
Published: June 2021

In contrast to adult, neonatal cardiomyocytes are able to proliferate and lose this capacity soon after birth when they withdraw from the cell cycle, become binucleated and differentiate. The arrest of cardiomyocytes cell cycle can be reversible for a short period, conferring the neonatal heart a regenerative potential within the first week of postnatal life. In the timeframe surrounding birth, heart maturation is also characterized by a change in energy metabolism, switching from glycolysis to beta-oxidation. However little is known about how metabolic programming in postnatal cardiomyocytes regulates their ability to proliferate, become binucleated and differentiate. In this study, we show that blocking beta-oxidation in mouse neonatal cardiomyocytes with etomoxir treatment promotes glycolysis and cell cycle re-entry, while increasing fatty acid beta-oxidation but reducing glycolysis leads to a decrease of the number of proliferating cardiomyocytes. In neonatal mice our data demonstrate that cardiomyocytes undergo binucleation and differentiation during the first week after birth and this process is correlated with the upregulation of the natriuretic peptides, ANP and BNP expression. Notably, in the postnatal mouse heart, beta-oxidation blockade through in vivo etomoxir injections, increases ventricular cardiomyocytes number, decreases natriuretic peptides expression and reduces the conversion of cardiomyocytes from a mononucleated to a binucleated phenotype. These findings highlight the importance of metabolic programming in regulating cardiomyocyte proliferation and suggest a potential therapeutic target for heart regeneration by modulating energy metabolic programming.
Abstract 68: p38 MAP Kinase Regulates Chamber Specific Postnatal Remodelling of Cardiac Ventricles
Authors: Yokota Tomohiro , Li Jin , Zhang Qing , Ding Yichen , Sung Kevin , Rau Christoph , Ren Vincent , Minamisawa Susumu , Hsiai Tzung , Kulkarni Rajan , Xiao Xinshu , Wang Yibin
Published: June 2021

Background:
Left ventricle (LV) and right ventricle (RV) in mouse heart undergo dramatically different chamber-specific remodeling after birth, leading to rapid increase in LV vs. RV chamber size. However, the underlying regulatory mechanism mediating chamber specific remodeling process remains enigmatic.

Results and Methods:
In neonatal mouse heart, p38 MAP kinase activity is dynamically activated in a chamber specific manner. p38 activity is specifically elevated in RV comparing to LV at E18.5, postnatal day 3 (P3) and P7 stages whereas p38 activity is lower in both ventricles at P0 and P1. In mouse heart with cardiomyocyte specific-knockout of p38α and β (p38ab-cdKO), total p38 activity was diminished in both chambers. The p38ab-cdKO mice had significant neonatal lethality associated with RV specific chamber enlargement and significant increase in both RV wall thickness (RVW) and inner diameter of RV (RVID) as early as P3. Interestingly, p38 inactivation suppressed myocyte apoptotic activity specifically in RV while increased RV myocyte proliferation and hypertrophy during neonatal period. Unexpectedly, RNA-seq results implicated Xbp1 mediated transcriptional regulation significantly contributing to p38 dependent transcriptome reprogramming in RV. Indeed, IRE1α expression in neonatal cardiomyocyte is sufficient to induce proliferation in vitro. Furthermore, knockdown of Xbp1 blunted p38 inhibition-induced myocyte proliferation, suggesting that IRE1a/Xbp1 mediate p38 signaling in neonatal myocyte proliferation.

Conclusion:
Chamber-specific remodeling in neonatal heart involves temporally regulated and RV specific p38 MAP kinase activity. RV specific myocyte proliferation and hypertrophy concurrent with RV specific programmed myocyte death is orchestrated by two innate stress-response pathways, p38 and Xbp1.
Abstract 69: Cardiomyocyte GSK-3α Signaling Exacerbate Pressure Overload-induced Dilated Cardiomyopathy and Heart Failure
Authors: Ahmad Firdos , Lal Hind , Verma Vipin K , Zhang Qinkun , Woodgett James R , Force Thomas L
Published: June 2021

Chronic pressure-overload (PO) induced-dilated cardiomyopathy (DCM) is one of the leading causes of left ventricular (LV) remodeling and heart failure. The role of glycogen synthase kinase-3α (GSK-3α) in PO-induced remodeling is not clear and existing dataset with global transgenic and knockout (KO) models show opposing roles. We sought to identify the specific role of GSK-3α in PO-induced dilatative cardiac remodeling. To better understand the role of GSK-3α, we employed cardiomyocyte-specific
GSK3A
(

GSK3A
fl/fl MerCreMer

) KO mice. Post-tamoxifen treatment, the GSK-3α KO and littermate control mice underwent sham or trans-aortic constriction (TAC) surgery. Heart function was assessed at 0, 2, 4 and 6 week post-TAC using serial M-mode echocardiography. Cardiac function in the KOs and littermate controls declines equally up to 2 weeks of TAC. At 4 week, KO hearts underwent further hypertrophy, retaining concentric LV remodeling and preserved contractile function both at systole and diastole. In contrast, wild-type LV showed significant chamber dilatation with an impaired contractility. Significantly reduced LV chamber dilatation [LVIDd(mm); 5.4±0.4 vs. 4.9±0.4,
P
=0.01] and preserved contractile function [LVEF(%); 22.2±12.6 vs. 40.0±18.7,
P
=0.02] remains same in the KO mice until the end of the study (6 wk). Furthermore, LV posterior wall thickness in the KO hearts, both at systole and diastole, were significantly greater in comparison to the controls. Consistent with preserved LV dimension, significantly less mortality was observed in the KO vs. control group during the remodeling phase. Histological analysis of heart sections further revealed better preserved LV chamber and protection against TAC-induced cellular hypertrophy in the GSK-3α KOs. Moreover, KO hearts showed significantly less fibrosis accompanied with low level of cardiomyocyte apoptosis post-6 wk of TAC. Taken together, these observations show that cardiomyocyte-specific deletion of GSK-3α protects against chronic PO-induced adverse LV remodeling and preserves contractile function. Inhibiting specifically GSK-3α using isoform-specific inhibitor could be a viable therapeutic strategy to limit the PO-induced DCM, adverse remodeling and heart failure.
Abstract 73: Yap Plays a Crucial Role in the Development of Cardiomyopathy in Lysosomal Storage Diseases
Authors: IKEDA SHOHEI , Shirakabe Akihiro , Sciarretta Sebastiano , Sadoshima Junichi
Published: June 2021

Rag proteins play a critical role in regulating lysosomal functions. Muscle-specific deletion of RagA and RagB (Rag A/B) results in lysosomal dysfunction, suppression of autophagy, and development of cardiomyopathy, reminiscent of lysosomal storage diseases (LSDs). We investigated the molecular mechanism by which cardiomyopathy is developed in RagA/B knockout (KO) mice. We generated cardiac-specific Rag A/BKO (RagA/BcKO) mice using αMHC-Cre. Similar to the muscle-specific KO mice, RagA/BcKO mice showed prominent cardiac hypertrophy (heart weight (HW)/ tibial length (TL): 12.35 ± 0.41 vs. 7.12 ± 0.15, p< 0.01) at 3 months of age. RagA/BcKO mice exhibited significantly greater phospho-histone H3-positive cardiomyocytes than control mice, suggesting that cardiomyocyte proliferation is stimulated in RagA/BcKO mice. However, RagA/BcKO mice exhibited a significantly smaller percentage of fractional shortening (%FS: 23.0 ± 0.82 vs. 45.8 ± 1.06, p< 0.01) and significantly greater mortality than control mice (50% vs. 0%, p<0.01). RagA/BcKO mouse hearts showed upregulation of β-MHC and α-smooth muscle actin, indicating de-differentiation. Yes-associated protein (YAP) is a transcription co-factor that controls growth and survival and is a key downstream target of the Hippo pathway. YAP associates with p62/SQSTM1 and is degraded through autophagy. YAP is significantly more accumulated (5.9-fold, p<0.05) in RagA/BcKO mouse hearts, where autophagy is inhibited and p62/SQSTM1 is accumulated, than in control hearts. In order to elucidate the role of YAP in mediating cardiomyopathy, we crossed RagA/BcKO mice with cardiac-specific heterozygous YAPKO mice. Heterozygous deletion of YAP in RagA/BcKO mice reduced the heart size (HW/ TL: 9.12 ± 0.10 vs. 11.9 ± 0.27, p< 0.05) and improved cardiac function (%FS: 35.0 ± 2.89 vs. 24.0 ± 0.58, p< 0.05) in RagA/BcKO mice. Pharmacological inhibition of YAP by verteporfin treatment also suppressed cardiac hypertrophy (HW/ TL: 8.92 ± 0.11 vs. 12.09 ± 0.30, p< 0.05) and heart failure (%FS: 33.3 ± 2.29 vs. 23.3 ± 0.63, p< 0.05) in RagA/BcKO mice. These results indicate that YAP plays a crucial role in the development of cardiomyopathy in RagA/BcKO mice, a mouse model of LSDs.
Abstract 98: Profiling GPCR Expression in Cardiac Fibroblasts and Myofibroblasts
Authors: Sriram Krishna , Aroonsakool Nakon , Michkov Alexander , Insel Paul
Published: June 2021

G-protein coupled receptors (GPCRs) are the largest class of cell surface receptors, serving as drug targets, at least in part, due to their diversity, selectivity in tissue expression and ability to regulate a wide variety of cellular functions. We hypothesized that cardiac fibroblasts (CFs) and pro-fibrotic myofibroblasts (myoFs) may express GPCRs that will regulate their activity and will identify previously unappreciated GPCRs as potential therapeutic targets for cardiac fibrosis. To test this hypothesis, we profiled non-chemosensory GPCR mRNA expression by using qPCR-based GPCR arrays in studies of CFs isolated from ventricular tissue of rats, mice and humans. Particular attention was paid to assessing cells at low passage (1-3) and grown on substrates that mimic the stiffness of cardiac tissue in-vivo. MyoFs were obtained by treating CFs ex-vivo with TGFβ, and from CFs that spontaneously transformed to MyoFs by growth on hard tissue culture substrates.We find that CFs from humans, rats and mice express ~120 GPCRs and that a majority of GPCRs (>75%), especially highly expressed GPCRs, are detected in human, rats and/or mice CFs; ~40% of highly expressed GPCRs are orphan receptors (without known physiologic agonists). Of GPCRs with known G-protein linkages, Gi-coupled receptors are most numerous, followed by Gq-, Gs- and G12/13-coupled GPCRs. GPCR expression profiles of rat atrial and ventricular CFs are highly similar in terms of both identity and level of expression. By contrast, GPCR expression in cardiac myocytes (CMs) differs significantly from CFs: most highly expressed receptors in CFs are undetected or much lower expressed in CMs. Several GPCRs detected in MyoFs have reduced expression vs CFs but a subset of GPCRs have higher expression in MyoFs. Validation of mRNA expression via protein detection as well as functional assays helps confirm the presence of a large number of the GPCRs.

Conclusions:
CFs and MyoFs from rodents and humans express ~120 GPCRs, including many orphan GPCRs. Atrial and ventricular CFs have similar GPCR profiles but ones that differ from that of CMs. CFs and MyoFs show differences in the number and nature of GPCRs expressed. We hypothesize that GPCRs higher expressed in MyoFs may contribute to their pro-fibrotic phenotype.
Abstract 107: Laser Angioscopy Reveals Concealed Ulcers in Substenotic Carotid Plaques in Cryptogenic Stroke Patients: A Preclinical Study
Authors: Savastano Luis E , Seibel Eric , Wang Thomas , Gordon David , Pandey Aditya S , Thompson Byron Gregory
Published: June 2021

Introduction:
Evidence suggests that a significant proportion of cryptogenic strokes may be due to artery-to-artery emboli from substenotic complicated carotid plaques. Thus, carotid artery disease not meeting etiological criteria for stroke (i.e. <50% stenosis) could still harbor thrombo-emboligenic lesions undetectable by standard imaging modalities. We developed an ultrathin, high-resolution, laser-based angioscope (4.7F, 200.000 pixels), capable of detecting such lesions.

Hypothesis:
We hypothesized that, unlike conventional imaging modalities, high-definition angioscopy would accurately detect small thrombi and ulcers in non-stenosing complicated atherosclerotic plaques.

Methods:
After institutional approval, the carotid arteries from 2 patients with cryptogenic strokes were harvested post-mortem and endoluminal surface videos were obtained with multimodal SFA. Angioscopic findings were compared to 25 carotid arteries autopsied from subjects with and without atherosclerosis, and 5 carotid endarterectomy (CEA) specimens from symptomatic patients. After angioscopy, all specimens were processed for histology and blindly reviewed by a pathologist; lesions were then matched to endoscopic findings. Subsequently, we tested the SFA in an
in vivo
rabbit model of atherosclerosis.

Results:
Punched-out lesions with slightly raised, irregular bead-like borders were encountered in the ipsilateral substenotic carotid arteries (<25%) of patients with cryptogenic stroke. This pathognomonic angioscopic architecture corresponded to ulcerated plaques, and was found exclusively in CEA specimens removed after stroke. As a prelude for clinical use, we demonstrated that SFA generates high quality images
in vivo
.

Conclusions:
Laser angioscopy reveals ulcerated plaques in carotid arteries with low-degree stenosis, obtained post-mortem from patients with cryptogenic strokes. Clinically, this novel platform could be used to identify ulcerated carotid plaques or intraluminal thrombi; such diagnostic resolution could help establish the etiology of a subset of cryptogenic strokes that present with low-degree carotid stenosis.
Abstract 11: Targeted Training of a Motor-parietal Circuit Improves Its Behavioral Output
Authors: Cramer Steven C , Zhou Robert , Ingemanson Morgan , Choi John J , Wu Katherine M , Kaur Arshdeep , Erani Fareshte , Yang Derek Z , Khaturi Nikhita , Cassidy Jessica M , Scacchi Walt , Dodakian Lucy , McKenzie Alison , Lopes Cristina V
Published: June 2021

Introduction:
Emerging brain mapping methods measure function of individual brain circuits and have the potential to predict a patient’s gains and needs in the context of stroke rehabilitation. We recently described a motor-parietal circuit underlying visuomotor tracking and defined an EEG coherence measure (reflecting connectivity) that predicts visuomotor learning. Here we test the hypothesis that this EEG metric predicts visuomotor learning after stroke.

Methods:
After baseline dense-array resting EEG, patients with chronic hemiparetic stroke were provided with a home-based gaming system. During 9 half-hour training sessions, patients played games in which the stroke-affected arm tracked objects moving on the tabletop. Games were implemented using augmented reality, which we have found has advantages for motor training and in which virtual objects are projected into the real world and modified during game play.

Results:
Subjects (n=12) had affected arm Box&Blocks score of 15±12 and were 35±26 mo post-stroke. Visuomotor tracking improved significantly: on a standardized visuomotor test using the gaming system, scores increased from 60.5±11.5% to 74.0±3.2% (p=0.003). Gains were specific, as other behaviors were unchanged. Individual gains in visuomotor tracking score were predicted by the EEG connectivity metric from our prior study, coherence between leads overlying ipsilesional primary motor cortex (M1i) and ipsilesional lateral parietal region in the high beta (20-30 Hz) range, with higher connectivity predicting greater visuomotor tracking gains (r=0.61, p=0.037). This too was specific, as connectivity between M1i and other brain areas did not predict gains. Secondary analysis found that baseline visuomotor tracking scores correlated with several EEG connectivity measures, all inversely and all between M1i and contralesional regions.

Conclusions:
We found that (1) training that targets a specific brain circuit improves behavioral output of that circuit, and (2) an EEG measure of brain connectivity within that circuit predicts these behavioral gains--both with specificity. This approach may be useful for many neural circuits and their respective rehabilitation-related behaviors.