bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 513: Emre Regulates the Mitochondrial Calcium Uniporter
in vivo
Authors: Liu Julia C , Syder Nicole , Ghorashi Nima , Willingham Thomas B , Parks Randi J , Sun Junhui , Fergusson Maria , Liu Jie , Holmstrom Kira M , Menazza Sara , Springer Danielle , Liu Chengyu , Glancy Brian , Finkel Toren , Murphy Elizabeth
Published: October 2020

Mitochondrial uptake of Ca
2+
plays critical roles in cardiac energy production as well as cell death. The mitochondrial calcium uniporter in mice and humans is a multi-protein complex that includes the channel-forming protein MCU and several other subunit proteins, including EMRE. EMRE is a single transmembrane protein that is conserved among metazoan species and is known to be essential for mitochondrial Ca
2+
uptake in cell culture. To investigate EMRE’s role in organismal physiology, we generated a mouse model of global germline EMRE deletion. We show that EMRE is indeed required for mitochondrial calcium uniporter function in isolated mitochondria from multiple tissues. Although the birth rate of

Emre
-/-

mice is lower than expected by Mendelian genetics (~5-10% instead of ~25%), the mice that are born are viable and appear healthy. Oxygen consumption in isolated mitochondria and cells is not significantly affected by loss of EMRE, and similarly the mice do not exhibit overt metabolic impairment, even under strenuous exercise. No significant differences between

Emre
-/-

and wild-type (
WT
) cardiac function at baseline and after isoproterenol stimulation are evident by echocardiography. Moreover,

Emre
-/

-
hearts are not protected from ischemia/reperfusion injury in a Langendorff perfusion model (mean infarct area 61% in

Emre
-/-

hearts; 57% in
WT
). Collectively, these data and their similarities to results found via germline
Mcu
deletion demonstrate that EMRE is indeed essential for mitochondrial Ca
2+
uptake in vivo. Furthermore, we find evidence that EMRE protein expression is elevated in some mouse muscular dystrophy models, suggesting that modulation of EMRE levels may play a role in regulating uniporter activity in conditions of stress or disease. We therefore further explore whether and how EMRE expression changes with isoproterenol-induced cardiac hypertrophy in mice and in samples from human patients with heart failure. Understanding of how uniporter components such as EMRE can regulate MCU in a diseased state can inform better therapeutic strategies aimed at restoring mitochondrial metabolic homeostasis.
Abstract 516: Homotypic Fusion Can Generate Bi- and Multi-nucleated Cardiomyocytes in the Postnatal Murine Heart
Authors: Ali Shah R , Menendez-Montes Ivan , Warshaw Jane , Xiao Feng , Sadek Hesham A
Published: October 2020

Multinucleation is an important and prominent phenotype of postnatal mammalian cardiomyocytes, and its occurrence in early life correlates with the loss of regenerative potential in mice. This phenomenon is believed to be the result of mitosis followed by cytokinesis failure, although direct proof of this mechanism has not been provided for every single adult cardiomyocyte. Moreover, many multi-nucleated cells in other organs are formed after homotypic cell fusion. Although heterotypic cell fusion between cardiomyocytes and circulating cells has been described as a rare event, it is not known whether homotypic myocyte fusion occurs and if it can lead to bi- and multi-nucleation. We developed a novel mouse model wherein multi-color fluorescent protein reporter expression occurs stochastically only in cardiomyocytes (Myh6-MerCreMer;Rosa26-Confetti). Using pulse-chase experiments during development and at birth, we show that a small fraction of cardiomyocytes fuses with other myocytes to generate binucleated and multinucleated cells; we calculate a fusion rate of at least 2%, although this reflects the lower limit of the phenomenon due to technical limitations of our model. We reveal that this process occurs in the first week of life and is stable at one month of age. Our findings suggest a revision of the dogma in postnatal cardiac development by providing another mechanism for multinucleation of cardiomyocytes. Future experiments will address whether fusion is a reversible process and whether fused myocytes can undergo abscission in response to physiological or pathological cues.
Abstract 517: Substrate Dependence of Mitochondrial Supercomplex Abundance in Murine Heart
Authors: Altamimi Tariq R , Audam Timothy N , Zheng Yuting , Gibb Andrew , Liu Siqi , Jones Steven P , Hill Bradford G
Published: October 2020

Mitochondrial supercomplexes are prominent in mammalian tissues that have high energy demand. Nevertheless, the mechanisms that regulate supercomplex formation and abundance remain unclear. In this study, we examined how myocardial fuel preference regulated by constitutive changes in phosphofructokinase (PFK) activity
in vivo
or by differential substrate provision to isolated mitochondria affect mitochondrial supercomplexes. Protein complexes from digitonin-solubilized cardiac mitochondria were resolved by blue-native polyacrylamide gel electrophoresis and were identified by mass spectrometry and immunoblotting to contain Complexes I, III, and IV as well as accessory proteins. Mitochondria from hearts with low PFK activity (Glyco
Lo
hearts) had higher mitochondrial supercomplex abundance and activity compared with mitochondria from wild-type (WT) or Glyco
Hi
hearts. Incubation of WT mitochondria with fatty acyl carnitine promoted higher supercomplex formation than did incubation with pyruvate, suggesting that substrate utilization is sufficient to regulate mitochondrial supercomplex abundance. These data are consistent with the hypothesis that mitochondrial supercomplex abundance is regulated in a substrate-dependent manner and suggest that metabolic scenarios favoring fat oxidation may promote supercomplex abundance.
Abstract 518: Phosphorylation of MK5 at Threonine-182 in the Activation Loop is Mediated by P38α/β in Cardiac Fibroblasts
Authors: Sahadevan Pramod , Nawaito Sherin A , Trepanier Joelle , Sahmi Fatiha , Villeneuve Louis , Gaestel Matthias , Allen Bruce G
Published: October 2020

MAP kinase-activated protein kinase-5 (MK5) is a protein serine/threonine kinase involved in fibroblast function. MK5 is activated by phosphorylation at threonine-182 (Thr182): p38α/β, ERK3, and ERK4 have been implicated. We examined the phosphorylation of MK5 in adult cardiac ventricular fibroblasts. In serum-starved cardiac myofibroblasts (fibroblasts maintained on a plastic substrate), phospho-MK5 Thr182 (pThr182) immunoreactivity was predominantly nuclear. In response to serum, sorbitol, angiotensinII, TGFβ, or H
2
O
2
, pThr182 immunoreactivity both increased in intensity and relocated to the cytoplasm and the perinuclear region. In each case, the p38α/β inhibitor, SB203580, prevented both the increase in intensity and redistribution of pThr182 immunoreactivity. Incontrast, siRNA-mediated knockdown of ERK3 resulted in a diffuse cytosolic distribution of pThr182 immunoreactivity but failed to attenuate the increase in intensity. On Phos-tag PAGE, the electrophoretic mobility of phosphorylated proteins is reduced. Phos-tag PAGE resolved MK5 immunoreactivity from actively dividing myofibroblasts into several slower-migrating bands that were absent following 1) pretreatment with phosphoprotein phosphatase or 2) including EDTA in the Phos-tag gels. In serum-stimulated myofibroblasts, SB203580 reduced both the abundance of lower-mobility forms of MK5 on Phos-tag PAGE and the abundance of MK5 immunoreactivity in ERK3 immunoprecipitates. When fibroblasts were maintained on a compliant (8-kPa) substrate, and hence quiescent, the lower mobility forms of MK5 immunoreactivity were less abundant relative to myofibroblasts. Furthermore, in whole heart lysates from micesacrificed 8 weeks after constriction of the transverse aorta (TAC), Phos-tag PAGE revealed banding patterns consistent with increased MK5 phosphorylation relative to sham hearts. Taken together, these observations suggest: 1) p38α/β are the primarymediators of MK5 phosphorylation at Thr182 in cardiac fibroblasts, 2) ERK3 may be responsible for targeting activated MK5 to specific cytosolic sites, 3) Thr182 is not the only site at which MK5 is phosphorylated
in vivo
, and 4) MK5 phosphorylation increases with fibroblast activation.
Abstract 520: Structural, Molecular, and Electrophysiological Maturation of Human Induced Pluripotent Stem Cell Derived Atrial Cardiomyocytes to Serve as a Model for Atrial Fibrillation
Authors: Ly Olivia T , Brown Grace , HONG Liang , Sridhar Arvind , Zhang Meihong , Chen Hanna , Youn Seock Won , Abdelhady khaled , Massad Malek , Alzahrani Zain , KHETANI SALMAN , Darbar Dawood
Published: October 2020

Background:
Atrial fibrillation (AF), the most common arrhythmia, is associated with significant morbidity and increased mortality. Although antiarrhythmic drugs are still commonly used to treat symptomatic AF, membrane-active drugs are incompletely and unpredictably effective, failing to target the underlying mechanisms of AF. Our pilot data shows atrial iPSC-CMs generated from a familial AF kindred recapitulated the electrophysiologic (EP) phenotype of an AF-linked SCN5A mutation, serving as a novel platform to target underlying cellular AF mechanisms. However, structural, molecular, and EP immaturity as compared to adult atrial CMs has hindered successful mechanistic evaluation.

Objective:
We aim to determine optimal condition(s) to enhance the maturity of atrial iPSC-CMs, establishing them as a novel platform to model AF, elucidate the cellular mechanisms, and identify/assess novel, mechanism based therapies.

Methods:
Maturity of atrial iPSC-CMs was enhanced using TID (T3, IGF-1, dexamethasone), fatty acids (FA), acute electrical stimulation (ES), and extracellular matrix (ECM) modulation. We examined for improvements in structural maturity (immunofluorescence, transmission electron microscopy), molecular (qPCR, RNA seq) and EP (patch clamping, multi-electrode array, high-throughput automated patch clamping). The maturity of atrial iPSC-CMs was compared with adult atrial CMs obtained from the same patient during cardiac surgery.

Results and Conclusion:
Fig. 1
demonstrates that acute ES, combined with TID and FA supplementation, significantly improves structural and in part EP maturity of atrial iPSC-CMs.
Abstract 521: Apolipoprotein M Links High-density Lipoprotein to Myocardial Autophagy
Authors: Javaheri Ali , Guo Zhen , Chandamarai Ezhil , Riehl Terrence , Valenzuela Ripoll Carla , Villa Torrecilla Juan Antonio , Rettig Michael , DiPersio John
Published: October 2020

Background:
Apolipoprotein M (APOM) is a lipoprotein that binds sphingosine-1-phosphate (S1P) and high-density lipoprotein (HDL). APOM/S1P attenuate myocardial ischemia/reperfusion injury by reducing infarct size. We recently published that reduced APOM plasma protein levels are associated with mortality across the spectrum of human heart failure. With the goal of uncovering mechanisms by which APOM impacts heart failure mortality, we modeled doxorubicin cardiotoxicity in hepatocyte specific APOM overexpression (APOM
TG
) and littermate controls.

Results:
Doxorubicin treatment acutely reduces APOM plasma protein levels in control mice. APOM
TG
mice exhibit ~3-5 fold increased APOM and ~2.5 fold increased S1P. In acute and chronic doxorubicin cardiotoxicity models, APOM
TG
mice were protected from mortality and reductions in left-ventricular mass and ejection fraction observed in littermate controls. In murine models of acute leukemia, doxorubicin effectively killed leukemic cells in control and APOM
TG
mice. In the myocardium, APOM
TG
mice and controls had similar levels of γ-H2AX foci after doxorubicin treatment, indicating similar levels of DNA damage, and electron microscopy indicated similar mitochondrial morphology. However, APOM
TG
mice exhibited enhanced autophagic flux, and APOM prevented doxorubicin-induced reductions in autophagic flux observed in littermate control mice (Figure). Mechanistically, treatment of neonatal rat cardiomyocytes with S1P mimetics attenuated doxorubicin-induced loss of lysosomal pH, suggesting that APOM may enhance lysosomal function via S1P. In the murine myocardium, doxorubicin reduced the nuclear protein content of transcription factor EB (TFEB), a master regulator of autophagy and lysosomal biogenesis, in control mice but not APOM
TG
mice. Furthermore, adeno-associated virus mediated knockdown of TFEB completely reversed the beneficial effects of APOM on the myocardium, leading to cardiomyopathy and mortality in APOM
TG
mice.

Conclusions:
Our studies identify APOM and S1P as regulators of TFEB in the myocardium, hence providing a mechanistic link between HDL and the autophagy-lysosome pathway in the murine heart. The role of APOM in myocardial autophagy may explain our observed clinical associations between reduced APOM and mortality.
Abstract 524: Phosphofructokinase Coordinates Anabolic Pathways in the Heart
Authors: Fulghum Kyle L , Lorkiewicz Pawel K , Cassel Teresa , Fan Teresa M , Jones Steven P , Hill Bradford G
Published: October 2020

Energy provision and biosynthesis are thought to be controlled by amphibolic enzymes such as phosphofructokinase (PFK). Nevertheless, it remains unclear how PFK influences collateral biosynthetic pathways in the heart. Here, we investigated the control exerted by PFK on anabolic pathways in the heart using
in vivo
deep network tracing. Wild-type (WT) mice and mice overexpressing kinase- or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgenes, termed respectively Glyco
Lo
or Glyco
Hi
mice, were fed a liquid diet containing
13
C
6
-glucose for 18 h. From freeze-clamped hearts, metabolite abundance and
13
C enrichment were then assessed by ion chromatography-mass spectrometry. Compared with Glyco
Hi
and WT hearts, Glyco
Lo
hearts had remarkably higher levels and
13
C enrichment of glucose 6-phosphate, fructose 6-phosphate (F6P) (p<0.01 each,
n
=4/gp), and purine and pyrimidine biosynthetic pathway intermediates. In addition to elevated activity of nucleotide biosynthetic pathways, we also found in Glyco
Lo
hearts higher
13
C-hexose incorporation into N-acetyl-D-glucosamine 6-phosphate (p<0.05); however, the levels of UDP-GlcNAc and its enrichment with
13
C were not different between the groups, which suggests that PFK activity influences the hexosamine biosynthetic pathway primarily by modulating F6P incorporation. Whereas Glyco
Lo
hearts showed lower
13
C fractional enrichment in triacylglycerols and hexosyl ceramide species, Glyco
Hi
hearts showed higher
13
C fractional enrichment in ceramide species. Interestingly, Glyco
Lo
hearts had 2-fold lower levels of alpha-ketoglutarate with concomitant increases in glutamate abundance. Energy charge values ranged from 0.69-0.81 with no differences observed between groups.These results suggest that PFK regulates lipid biosynthesis and collateral biosynthetic pathways of glucose metabolism in the heart. Understanding how these pathways contribute to cardiac remodeling could be important for developing metabolic therapies to improve cardiac health.
Abstract 527: Role of IGFBP3 in Neonatal Heart Regeneration
Authors: Ali Shah R , El-Helaly Waleed , Menendez-Montes Ivan , Nguyen Ngoc Uyen Nhi , Thet Suwannee , Sadek Hesham A
Published: October 2020

Background:
The adult mammalian heart is unable to regenerate after an injury. However, newborn mice are able to fully regenerate the heart after myocardial infarction (MI). The neonatal MI model therefore is a potential blueprint for regenerating the adult heart that could offer novel therapies for patients suffering from heart disease. To investigate the mechanism by which neonatal heart regeneration occurs, we screened for secreted proteins that are upregulated after neonatal MI but not after adult MI. We hypothesized that such a protein could be a cardiomyocyte mitogen that underlies the cardiomyocyte proliferation that occurs after neonatal MI.

Methods:
We performed microarrays on neonatal and adult MI (and sham) heart tissue samples: we identified IGFBP3 (Insulin Growth Factor Binding Protein 3), which canonically transports IGF ligands in circulation, as a secreted protein that is uniquely upregulated after neonatal MI. We used in situ hybridization, reporter mice, and immunostaining to validate the findings from the microarray. Single cell RNA-seq data revealed that IGFBP3 is expressed in vascular cells.

Results:
We first tested whether IGFBP3 is necessary during neonatal heart regeneration: we performed neonatal (P1) MI in global Igfbp3 knockout mice and wild-type mice, and found that knockout mice have more fibrosis and worse ejection fraction (EF) one month after P1 MI. To determine if IGFBP3 is sufficient to promote cardiomyocyte proliferation, we injected recombinant IGFBP3 protein into the heart of one week-old mice (P7) and saw more cycling myocytes. We generated novel transgenic vascular-Igfbp3-overexpression mice, which exhibit less fibrosis as well as improved EF after P7 MI compared to controls.

Conclusions:
IGFBP3 is a secreted protein that is necessary for complete regeneration after a neonatal MI. Its ectopic expression can cause cardiomyocyte proliferation and can improve systolic function, and it prolongs the window of neonatal heart regeneration. Therefore, IGFBP3 may represent a cardiomyocyte mitogen with potential therapeutic value for adult heart disease.
Abstract 530: Mechanisms Underlying Phospholamban L39 Stop (PLN L39X) Cardiomyopathy
Authors: Bailey Rasheed A , Stillitano Francesca , Turnbull Irene , Haghigi Kobra , Fish Kenneth , Akar Fadi , Dubois Nicole , Wickramasinghe Nadeera , Hajjar Roger J , GELB Bruce D , Costa Kevin D , Kranias Evangelia , Triveri MariaGiovanna
Published: October 2020

Background:
Defective calcium (Ca++) handling is a hallmark of HF across species. Together with the Sarco/Endoplasmic Reticulum Ca-ATPase (SERCA2a), Phospholamban (PLN) has emerged as a critical regulator of Ca++homeostasis. Worldwide, PLN mutations are identified with increasing frequency in patients with dilated, hypertrophic and arrhythmogenic cardiomyopathy (CMPs) but the causative defects leading to the CMP remain incompletely understood. While preclinical studies have unequivocally shown that absence of PLN (PLNKO) is therapeutic in rodent HF models, the discovery of a human pathogenic mutation (L39X) presumed to be the human equivalent of the PLNKO lead to the conclusion that PLN ablation was lethal in human, mitigating any enthusiasm in targeting PLN inhibition in PLN-associated disease or HF treatment. The objective of this proposal is to levarge the use of “induced-pluripotent” stem cells (iPSCs) derived cardiomyocytes (CM) from homozygote L39X carriers to elucidate the role of PLN (L39) in human pathophysiology.

Methodology and Results:
We obtained mononuclear cells from Homozygotes (Hom) L39 carriers and generated 11 iPSC clones. To derive CMs, we used the direct differentiation method temporally modulating the Wnt/β-catenin signaling. Immunocytostaining revealed positive expression of cardiac troponin T as well as PLN. In Hom L39 derived CMs, PLN showed an abnormal cytoplasmic distribution, formed intracellular aggregates and there was loss of perinuclear localization when compared to matched WT iPSC-CMs. Using fura-2AM, we observed decreased calcium transient amplitude in iPSC-CMs from L39 compared to WT with prolongation of the time constant of relaxation and early after depolarization (EAD). Lastly, we saw a 70% and 50% reduction in the protein and mRNA expression of PLN and SERCA2a respectively.

Conclusions:
Our data suggest that the L39 PLN mutant is expressed but mis-located within the cardiomyocytes. The mis-location of PLN was associated to decreased SERCA2a expression impaired Ca++ handling and increased arrhythmogenicity.
Further studies will be required to fully elucidate the impact of the mutation in HF pathophysiology
Abstract 535: Mathematical Modeling of Hyperpolarized Pyruvate Metabolism in Human Heart
Authors: Yaros Katarina , Park Jae Mo , Malloy Craig , Alger Jeffry , Hussain Tarique , Zaha Vlad G
Published: October 2020

Background:
Dissolution dynamic nuclear polarization is a novel method that increases more than 10,000-fold the
13
C signal-to-noise ratio and emerges as a useful tool for detection of molecules much less prevalent than water in the human body. Hyperpolarized [1-
13
C] pyruvate (HP) permits the noninvasive, nonradioactive study of metabolic flux via pyruvate dehydrogenase, a key enzyme in the complex cardiac adaptation to physical activity, energy sources, and in various disease states. The aim of this study is to define an analysis pipeline of multiparametric, non-steady-state, real-time human cardiac magnetic resonance spectroscopic investigations.

Methods:
HP was infused intravenously into five human subjects. Dynamic spectroscopic data was acquired for [1-
13
C]- pyruvate,
13
C-alanine, [1-
13
C]-lactate, and
13
C-bicarbonate at two consecutive times for each subject. Imaging data were exported into MATLAB. We employed a first-order kinetic model to fit the signal for the hyperpolarized metabolites assuming bidirectional flow between pyruvate and lactate as well as pyruvate and alanine. A gamma variate input function was used to model the initial pyruvate bolus. We also calculated non-parametric values to describe the signal for each metabolite, specifically area-under-curve (AUC) and time-to-peak (TTP). The repeatability analysis was performed using an ANOVA-based method.

Results:
Rate constants derived from the kinetic model: kPL 0.016 ± 0.008s
-1
, kPA 0.012 ± 0.006s
-1
, kPB 0.024±0.0095s
-1
are of similar order of magnitude to prior studies. The reverse constant for lactate to pyruvate conversion (kLP) is relatively unchanged across the subjects (0.0099±0.0005), and reverse constant for alanine to pyruvate (kAP) conversion is almost negligible (0.0008 ± 0.0012). The model well describes the metabolites signal over time. Within subject repeatability was higher for TTP (lactate: 0.981 ± 0.018) ) than for rate constants (kPL 0.823 ± 0.153) and AUC (lactate 0.697 ± 0.244) with kPB having the lowest repeatability (0.325 ± 0.424) due to a single outlier.

Conclusions:
We present a real-time pipeline for analysis and modelling of pyruvate metabolism in the human heart and confirm that it is reproducible within the same subject under same conditions.
Abstract 537: Combination of Mitochondrial and Cytosolic Antioxidant Upregulated MSC Therapy Improves Non-Alcoholic Fatty Liver Disease (NAFLD)
Authors: Nandula Seshagiri Rao , Aimalla Nikhila , KUNDU Nabanita , Sen Sabyasachi
Published: October 2020

Introduction:
Diabetes and obesity are known to cause adipose inflammation, elevated oxidative stress by reactive oxygen species (ROS) accumulation. In this report we studied the effect of human adipose derived mesenchymal stroma cells (MSCs) overexpressing antioxidants both mitochondrial (Sod2) and cytosolic (Catalase) to reduce oxidative stress and systemic inflammation in DIO mouse model.

Methods:
C57BL/6J male mice (4–6 weeks old) were obtained from the Jackson Lab. Obesity, glucose intolerance, and insulin resistance were induced by feeding the mice a 60% fat high-fat diet (HFD) for 12 weeks. Mouse adipose-derived MSCs and adenovirus constructs containing GFP (Null, SOD2 and Catalase), were obtained. Liver and fat tissues gene expression, liver and fat depots H& E staining and liver triglycerides (L-TG) were quantified.

Results:
Triglyceride assay confirmed reduced liver fat accumulation in animals that received combination -MSCs. The gene expression analysis by qPCR for liver and omental fat shows upregulation of Ucp1, Pgc1a and Prdm16 genes with concomitant increase in all antioxidant (Sod2, Sod3 and Catalase) mRNA expression. Further the systemic TNF-α levels were decreased in the mice received MSC-Sod2+catalase. Glucose tolerance showed a improvement trend with low area under curve (AUC) values for mice that received MSCs with both Sod2 and catalase upregulated compared to control null group.

Discussion:
The combination effect of both mitochondrial and cytosolic anti-oxidants reduced systemic inflammation and liver triglyceride levels in diet induced obesity mouse model compared to SOD2, Catalase and Null MSCs. An increase mRNA expression of genes associated with browning of white adipose tissue deposits might have contributed to improvement in NAFLD along with systemic reduction in TNF-α. Genetically modified MSC therapy can be a promising option for treating obesity and diabetes.
Abstract 539: Buthionine Sulfoximine (BSO) Prevents Reductive Stress-induced Pathological Hypertrophic Cardiomyopathy
Authors: Shanmugam Gobinath , Litovsky Silvio , Pogwizd Steven M , Rajasekaran Namakkal S.
Published: October 2020

Background:
Chronic reductive stress (RS) induces pathological cardiac remodeling and diastolic dysfunction. Here, we hypothesized that preventing RS via glutathione (GSH) depletion, through selective inhibition of γ-glutamyl cysteine ligase (γGCL), mitigates cardiac pathology in cRS mice.

Methods:
Cardiac-specific constitutively active Nrf2 TG-mice (α-MHc-caNrf2-TG), at 6 weeks of age, were administered with buthionine sulfoximine (BSO; 5.0 mM/Kg; daily for 16-weeks). At the end of 22 weeks, cardiac structure and function (systole & diastole using echocardiography), myocardial redox state, levels of ROS (using dihydroethidium (DHE) fluorescence), and antioxidant proteome were assessed in TG mice treated with PBS or BSO and compared with the age matched NTG littermates (n=6/group).

Results:
While the TG mice experiencing RS (GSH; 426.3±22.55 vs. 141.8±3.9 & GSH/GSSG; 61.04±5.4 vs. 23.27±1.3 in TG vs. NTG), this was significantly curtailed in BSO-treated TG mice (GSH 161.8±12.6 & GSH/GSSG; 25.1±4.3). This was coupled with the normal cardiac functions (EF; ~53% & MV E/A; 1.57) in the BSO-treated TG when compared to TG mice experiencing a hyper systolic function (>80% ejection fraction) with decreased cardiac volume and diastolic dysfunction with restricted filling (MV E/A ratio; >3.0), Of note, BSO treatment did not alter the protein levels of antioxidants (i.e. GCLC, GCLM, NQO1 and CAT), but only depleted the GSH pool (via inhibiting GCL activity) and restored the basal ROS signaling in the myocardium. Moreover, the BSO-treated TG mice did not develop cardiac hypertrophy, which was assessed by heart weight/body weight ratio, qPCR-based gene expression for hypertrophy (
Anf, Bnf, α-MHc and β-Mhc
).

Conclusion:
Our results suggest that pharmacological manipulation of myocardial redox and repletion of basal ROS signaling prevented RS-mediated pathological processes and rescued the cardiac structure and function.
Abstract 540: Effects of Pro-inflammatory High Density Lipoprotein on Nrf2 Signalling and Foam Cell Formation in Macrophage of Coronary Artery Disease Patients
Authors: sunny sini , Narayani Jayakumari , Devarajan Asokan , S. Rajasekaran Namakkal
Published: October 2020

Background:
High density lipoprotein (HDL) from coronary artery disease (CAD) patients display proinflammatory properties, which persuades LDL oxidation and foam cell formation in monocytes/macrophages (Mo/MΦ). Nrf2/antioxidant signalling are crucial for maintaining the redox milieu, but these mechanisms are remain elusive in the setting of pro-inflammatory HDL (
pi
HDL) mediated atherosclerosis and coronary artery disease (CAD).

Methods:
In healthy humans (n=20) and CAD patients (n=20), we assessed Nrf2 signalling, paraoxonase-1 activity, HDL function and foam cell formation in the circulating Mo/MΦ.

Results:
We observed a range of HDL activity (20-80%) in healthy humans, indicating that the HDL function varies among normal individuals. In comparison with functional HDL (
f
HDL),
pi
HDL demonstrated decreased paraoxonase-1 activity (41.38±6.71
vs.
118.04±11.18; p<0.05), higher levels of lipid peroxides (0.95±0.09
vs.
0.02±0.01; p<0.05), higher triglyceride (75.4±6.79
vs.
31.76±4.19; p<0.05) and phospholipid levels (74.09±5.9
vs.
46.47±6.11; p<0.05) in CAD patients
vs
. healthy humans. Interestingly, we noticed an increase in MMP9 along with enhanced
pi
HDL in CAD patients, a previously unrecognized event, which might accelerate atherosclerosis in these individuals. Despite an increased Nrf2 nuclear translocation, we noticed significant decrease in the levels of antioxidant enzymes in the macrophages treated with
pi
HDL. It is likely that
pi
HDL mediated Nrf2 activation could be a compensatory response to ROS/oxidative stress in CAD patients, but insufficient antioxidant system might have resulted in enhanced foam cell formation.

Conclusions:
Taken together, these results suggest that
pi
HDL could be a key regulator of Nrf2 signalling in the Mo/MΦ of CAD patients. Thus, an early screening and identification of dysfunctional HDL (i.e.
pi
HDL) is crucial for vascular health, thereby preventing the progression of CAD.
Abstract 543: Clinical Development of Cardiac Reprogramming Gene Therapy
Authors: Zhou Huanyu , Lombardi Laura M , Reid Christopher A , Yang Jin , Srinath Chetan , Zeng Aliya , Leon Elena C , Qureshi Tawny N , Nettesheim Emily R , Xu Emma , Duclos Zoe , Liu Jun , Jones Samantha , Feathers Charles , Chung Tae Won , Jing Frank , Prince William , Lin JianMin , Hoey Timothy , Ivey Kathryn
Published: October 2020

Heart failure affects an estimated 38 million people worldwide and is typically caused by cardiomyocyte (CM) loss or dysfunction. Although CMs have limited ability to regenerate, a large pool of non-myocytes, including cardiac fibroblasts (CFs), exist in the postnatal heart. In vivo reprogramming of non-myocytes into functional CMs is emerging as a potential new approach to treat heart failure and substantial proof-of-concept has been achieved in this new field. However, challenges remain in terms of clinical application. First, reported human reprogramming cocktails often consist of five to seven factors that require multiple AAV vectors for delivery. Thus, a less complex cocktail that is able to fit into one AAV vector is needed for this technology to impact human health. Second, the lack of specificity in AAV tropism further complicates the safety and regulatory landscape. A means to limit the expression of reprogramming factors to target cells is critical for maximizing long-term safety. Lastly, although promising studies in small animals have already been reported, safety and efficacy results in large animal MI models are critical to justify cardiac reprogramming in human clinical trials.

We have developed a novel human cardiac reprogramming cocktail that consists of only two transcription factors and one miRNA. This new cocktail has been engineered into a single AAV cassette to efficiently reprogram human CFs into cardiomyocytes. We also substantially improved transduction of hCFs through AAV capsid engineering and eliminated CMs expression through a microRNA de-targeting method. Moreover, our novel cardiac reprogramming gene therapy improved cardiac function in both rat and swine MI models upon delivery at various time-points after MI without inducing arrhythmias. Given these promising safety and efficacy results in larger animals, we endeavor to translate direct cardiac reprogramming for clinical application.
Abstract 544: Increase in Isoflurane-mediated Blood-brain-barrier (bbb) Permeability is Regulated by Mmp3 via the Erk Signal Pathway
Authors: Wu Rongxue , Zheng Mei , Liu Lifeng , Betancourt Cristian , Sitikov Albert , Zhao Qiong , Liao James K , Wu Rongxue
Published: October 2020

Introduction:
The blood-brain barrier (BBB) normally maintains the CNS microenvironment. The effect of isoflurane, widely used in clinical anesthesia, on the BBB permeability has not been well studied. Matrix metalloproteinase 3 (MMP3) is implicated in the disruption of BBB. However, the direct role for MMP3 in regulating BBB and the underlying molecular mechanism has not yet been elucidated. We hypothesized that MMP3 plays a critical role in isoflurane mediated increase in BBB permeability.

Methods:
We used MMP3 deficient (MMP3
-/-
) mice and its littermate, wild-type (MMP3
+/+
), as a control. Animals were assigned to either vehicle-air or isoflurane in 3 different groups: control, MMP3
-/-
and control+MMP3 administration. We evaluated the anesthetic effect of isoflurane by the time of anesthesia induction, emergency time and volume of isoflurane. MMP3 levels were measured in different organs. We monitored the permeability of mouse BBB through in vivo (Evans blue dye extravasation, Sodium-FITC, and brain water content) and an in vitro model of BBB (Electrical cell impedance sensor assay and transwell permeability assay). To determine the mechanism of MMP3 on BBB permeability, we also used an ERK-inhibitor. Expression of the endothelial tight junctional protein was detected by both western blot and immunofluorescence staining.

Results:
The use of MMP3 increased the anesthetic sensitivity of isoflurane compared with the control group. In contrast, MMP3 KO mice displayed significantly longer induction time, higher isoflurane usage volume, and lower emergency time. Data showed that MMP3 increased BBB permeability in both in vivo and in vitro experiments, which is associated with a reduction in occluding, ZO-1 and claudin-5. We found mmp3 levels had negative correlations with tight junctional proteins and positive associations with p-ERK. Moreover, the use of an ERK-inhibitor abolished the effect of MMP3 on the junctional protein and in vitro BBB permeability.

Conclusion:
Our data suggest that MMP3 is required for modulation of isoflurane mediated BBB permeability by the reduction of tight junctional protein expression via the ERK pathway. Thus, mmp3 enhances the anesthetic effect of isoflurane, while the deletion of MMP3 protects against BBB disruption.
Abstract 545: Sex Difference in Right Ventricular Response to Pressure Overload is Associated With Differential Fibrotic Remodeling and Cyclic Guanosine Monophosphate Signaling
Authors: Russo Ilaria , Dun Wen , Mehta Swasti , Tzimas Christos , Tsai Emily J
Published: October 2020

Background:
In HFpEF and PAH, men are more likely than women to develop right ventricular dysfunction (RVD) and die. Mechanisms underlying these sex differences are not well understood. In advanced HF patients, RVD is associated with RV fibrosis. Sex differences in cGMP anti-fibrotic cardioprotection have been reported.

Hypothesis:
Sex differences in RV response to pressure overload may be due to differential anti-fibrotic cGMP signaling in the RV.

Methods:
Adult male and female C57BL6/J mice underwent pulmonary artery banding (PAB, 25g) vs. thoracotomy (Sham). RV remodeling was assessed by echocardiography, pressure-volume loop recordings, morphometrics, and histology. Expression of transcripts encoding various cGMP signaling proteins (
Nppa, Nppb, Npr1, Npr2, Npr3, Gucy1a3, Gucy1b3, Pde5, Pde9a, Pkg1a
), the fetal gene program, tissue fibrosis and ECM remodeling (
Ace, Tgfβs, Smads, Ctgf, αSma, Postn, Col1α1, Lum, Igfbp5, Mmp2-3, Timp1-2)
were assessed at 9wks.

Results:
Post-PAB survival rate did not differ between sexes (log-rank HR 1.02, 95% CI 0.30-3.48, P=NS), but median time-to-death was 20 days (IQR 15-32) for M-PAB vs. 35.5 days (IQR 10.8-41.5) for F-PAB. PAB increased RV mass in both sexes (Fulton’s index 0.45±0.07 M-PAB vs 0.21±0.01 M-Sham, P<0.01; 0.49±0.06 F-PAB vs 0.22±0.01 F-Sham, P<0.01). RV myocyte hypertrophy was greater in M-PAB (cardiomyocyte area 592.7±6μm
2
vs 397.7±21.6μm
2
F-PAB, P<0.01). RV dilation and decreased TAPSE and RV S’ were present in all PAB mice. Only M-PAB had depressed RV FAC (29.9±1.9% vs. 38.1±2.4% F-PAB, P=0.02). M-PAB had worse diastolic function than F-PAB (Tau 32.8±2.9ms M-PAB vs 14.2±2.1ms M-Sham, P<0.01; 25.6±5.3ms F-PAB vs 15.0±2.1ms M-Sham, P=NS). In male but not female mice, PAB induced upregulation of
Nppa, Nppb, Npr1, Npr2, Npr3, Gucy1a3, Gucy1b3, Pde5 and Pkg1a
in the RV. RV transcript levels of
Ace, Col1α1, Ctgf, Igfbp5, Mmp2, and Timp2
were also higher in M-PAB than F-PAB.

Conclusions:
Males have worse RV functional response to PAB than do females. This sex difference was also associated with increased mRNA levels of cGMP signaling mediators and upregulation of pro-hypertrophic and pro-fibrotic genes in male PAB mice. Further studies are needed to elucidate sex-dependent regulation of RV remodeling.
Abstract 548: Cellular Contributions to Neonatal Cardiac Regeneration
Authors: Misra Adwiteeya , Quijada Pearl J , Burke Ryan , Dirkx Ronald , Small Eric M
Published: October 2020

While the neonatal mouse heart possesses a remarkable ability to regenerate up to a week after birth, the adult mammalian heart is susceptible to irreversible scar formation that impedes cardiac function. Such a scar is formed by the precocious deposition of extracellular matrix (ECM) by resident cardiac fibroblasts (CFs). Unlike their adult counterparts, neonatal CFs in the regenerative widow may have a unique phenotype that contributes to cardiac regeneration and scar resolution. Indeed, the neonatal cardiac ECM secreted by CFs is reported to stimulate regeneration, yet the underlying mechanisms of CF-mediated cardiac repair in the neonate has not been examined. Here, we present a strategy to establish the role of tissue resident CFs in mouse neonatal cardiac regeneration through selective cell depletion and RNA sequencing. Through the initial analysis of published RNA sequencing data, we identified an enrichment of pro-regenerative molecules such as amphiregulin (Areg) during the neonatal regenerative window. Areg, an epidermal growth factor ligand, has been shown to paradoxically stimulate both cardiac repair and pathological fibrosis after adult cardiac injury. To assess its impact on the neonatal cardiac injury response, we developed an adeno-associated virus serotype 9 with the complete coding sequence of mouse Areg (AAV9:Areg) controlled by the cardiomyocyte-specific cardiac troponin T promoter.
In vivo
, AAV9:Areg treated mice accumulate BrdU+ (proliferative) non-myocytes adjacent to Areg-expressing cardiomyocytes. Ongoing studies are aimed at evaluating the contribution of CFs to neonatal cardiac repair, including whether Areg-dependent cellular changes impact CFs in the neonatal regenerative window in a manner distinct from that in the adult injury response.
Abstract 549: Characterization of Mesenchymal Stromal Cell-derived Exosomes From Patients With Improved Function Outcome After Ischemic Cardiomyopathy: A Biorepository Evaluation of the Focus-CCTRN Trial
Authors: Chacon Lourdes I , Mesquita Fernanda , Virk Hassan , Hochman-mendez Camila , Taylor Doris A
Published: October 2020

Mesenchymal stromal cells derived from bone marrow (BM-MSCs) have been considered promisingcandidates for regenerative medicine therapies for more than 20 years, but to date cardiovascular clinicaltrials of MSC-based therapies have demonstrated only modest improvement. However, the mechanismsunderlying why some patients improve and others do not remain undefined. Although MSC survival aftertransplantation remains a challenge, several studies have reported that BM-MSC-mediated paracrineeffects and delivery of exosomes may be therapeutically relevant. We hypothesized that MSC-derivedexosomes from patients with cardiac functional improvement contain factors that promote cell survival,proliferation, and cardiac protection. Here, we compared exosomes (derived from MSCs that had beenexpanded from BM-MNCs used for autologous transplantation) from two cohorts of disease- and age-matched consented patients with ischemic cardiomyopathy enrolled in the FOCUS-CCTRN trial—thosewith improved (cohort1, n=3) or worsened (cohort2, n=3) functional outcome in three primary endpoints:left ventricular (LV) ejection fraction, LV end-systolic volume, and maximal oxygen consumption.MicroRNA-seq analysis identified 16 upregulated and 12 downregulated miRNAs in cohort 1 whencompared to cohort 2. Using DIANA-Mir Path v.3.0, we identified 3,522 experimentally validated genetargets. Gene set enrichment analysis identified enrichment for fatty acid biosynthesis and metabolism,Hippo signaling pathway, protein processing in endoplasmic reticulum, adherent junction, cell cycle,endocytosis, RNA transport and degradation, estrogen-signaling pathway, regulation of actincytoskeleton, Prolactin signaling pathway, Focal adhesion, Notch-signaling pathway, p53-signalingpathway, insulin-signaling pathway and HIF-1-signaling pathway in the targets of upregulated miRNAs.The downregulated miRNAs were related to extracellular matrix-receptor interaction, protein processingin endoplasmic reticulum, cytokine-cytokine receptor interaction and antigen processing andpresentation. Thus, the identified miRNAs differentially expressed in patients with an improved functionaloutcome that may play a role in the molecular mechanisms that underlie in cardiac repair in patients withischemic cardiac disease. Moreover, these finding indicate that not all autologous cell products may becompetent therapy candidates for cardiac repair These results warrant further investigation to confirmthe effects of the identified differences on the target genes, which could improve the prognosis and unveilnew therapeutic approaches.
Abstract 551: Distinct Transcriptional Roles for Wildtype vs. Truncated Nrf2 in the Heart of Transgenic Mice
Authors: sunny sini , Crossman David , Shanmugam Gobinath , S. Rajasekaran Namakkal
Published: October 2020

Background:
Nuclear erythroid-2 like factor-2 (Nrf2), a redox-sensitive transcriptional regulator of cytoprotective and antioxidant genes. Despite the basal transcription of antioxidants/redox genes, forced activation of Nrf2 may lead to “reductive stress” (RS). In this study, we hypothesized that controlled vs. sustained activation of Nrf2 differentially regulates the myocardial transcriptome, which elucidates the transition of a physiological adaptation to pathological process.

Methods:
Cardiac specific Nrf2 transgenic mice expressing full length Nrf2 (FL-TG) and truncated Nrf2 (TR-TG), and their non-transgenic littermates (NTG) in the C57/BL6J backround at the age of 6-8 months were used for this study. Next generation RNA sequencing was performed using the myocardial mRNA obtained from FL-TG, TR-TG and NTG mice (n=3/group). Validation of the NGS data was carried out by qPCR analysis using specific primers for the targeted genes(n=4 to 6/group).

Results:
Analysis of the NGS data uncovered eccentric genomic profiles in the myocardium of mice with a 310 differentially regulated genes (DEGs) in FL-TG (
vs.
NTG) and 472 DEGs in TR-TG (
vs.
NTG). A closer evaluation reveals 397 and 237 DEGs commonly shared between FL-TG and TR-TG, respectively. STRING analysis revealed genomic networks involve in oxidoreductase, antioxidant and glutathione transferase were differentially altered between TL-TG vs. TR-TG groups. For instance, within the glutathione synthesis pathway,
Gclm
transcript was upregulated 6.0 fold (FL-TG) and 8.0 fold (TR-TG) when compared to NTG. Indeed, a significant douwnregulation of stress response genes
Hspa1b
(5.0 & 2.0 fold)
and Hsph1
(2.0 & 2.2 fold) was apparent in between the groups. An eccentric cytoskeletal disarrangement (
Efnb3, Map1a, Acta
), an impaired protein quality control (
Bin1,Myl4
) and a reduced cardiac function (
Timp4)
were significantly downregulated in both the groups.

Conclusion:
These findings conclude that transgenic expression of wild-type vs. mutant forms of Nrf2 in the heart uniquely regulate the myocardial transcriptome, which might result in independent pathological processes.
Abstract MP103: Methylglyoxal Modifications on Myofilament Proteins Caused by Diabetes Block Heart Failure Drug Targets Through Changing Tropomyosin Position
Authors: Papadaki Maria , Kampaengsri Theerachat , Barrick Samantha , Campbell Stuart , Greenberg Michael , Kirk Jonathan A
Published: October 2020

We previously discovered methylglyoxal (MG), a glycolysis byproduct, modifies arginine and lysine residues on myofilament proteins to a greater extent in patients with diabetes and heart failure than non-failing controls. These modifications reduced myofilament calcium sensitivity (pCa
50
), maximal calcium activated force (F
max
) and cross-bridge cycling dynamics. We hypothesized that compensating for these modifications might slow or halt the development of diabetic cardiomyopathy. We attempted to pharmacologically compensate for the negative functional effects of MG using omecamtiv mecarbil (OM), a myosin activator currently in clinical trials. As reported previously, we found that OM increased pCa
50
and decreased F
max
in normal myocytes. However, in myocytes pretreated with MG, OM had no effect of pCa
50
but still decreased F
max
. These data suggest OM may have less, or potentially detrimental effects in diabetic patients.To understand why MG might block the actions of sarcomere drug targets, we aimed to further elucidate the molecular mechanisms of MG. We utilized a computer model of sarcomere contraction to predict what molecular effects of MG would explain the functional data we collected. The model predicted a simultaneous increase in cross-bridge detachment rate and a decrease in the closed-to-blocked tropomyosin transition rate could explain the data. Stopped flow experiments measuring myofilament kinetics indicated a decreased transition rate between “closed” and “blocked” tropomyosin state but did not indicate a difference in the cross-bridge detachment rate upon treatment with MG. This finding further explained the observation that MG blocks OM’s effects - since a myosin activator would not necessarily be effective if MG affected tropomyosin position on actin. We hypothesize that a residue we found to be modified by MG on actin, K291 would be important for the molecular mechanism observed, since it is located in a critical position for the interaction between actin and tropomyosin. In this study, we have elucidated the mechanism of MG’s detrimental myofilament impact and found that that these modifications reduce the efficacy of OM. This mechanistic understanding will help drive future identification of therapeutic targets.