bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 397: Preventing Cardiomyopathy of Muscular Dystrophy Through Antagonism of the Thromboxane/Prostanoid Receptor
Authors: Carrier Erica J , Galindo Cristi L , Kim Kyungsoo , Macias-Perez Ines , Pavliv Leo , Shin Jonghyun J , Knollmann Bjorn C , Soslow Jonathan H , Markham Larry W , West James D
Published: February 2019

Muscular dystrophy causes mechanical damage and increased membrane permeability of cardiomyocytes, leading to progressive cardiomyopathy and diffuse myocardial fibrosis that typically begins in the free wall of the LV. Cardiac dysfunction is a primary cause of death in Duchenne (DMD) and other muscular dystrophy (MD) patients. Activation of the thromboxane/prostanoid (TP) receptor increases calcium transients in cardiomyocytes, causes arrhythmia, and is pro-fibrotic. We thus hypothesized that TP receptor activation contributes to the cardiac phenotype of MD, and that blockade of the TP receptor would improve cardiac fibrosis and function in mouse MD models. We gave 3 different mouse models of MD either normal drinking water or water containing 25 mg/kg/day of the TP receptor antagonist ifetroban, from weaning to the predetermined endpoint. TP receptor antagonism improved 10-week survival from 60% to 100% in utrophin/dystrophin double knockout mice, a model of severe DMD, and increased cardiac output compared with surviving vehicle-treated mice. In the mdx/mTR mouse model of DMD, treatment with ifetroban likewise improved 6-month survival from 43% to 100% and increased cardiac output. Finally, we examined delta-sarcoglycan knockout (dSG KO) male mice, a model of limb-girdle muscular dystrophy (LGMD) that replicates the DMD cardiac phenotype with improved survival. TP receptor antagonism normalized fractional shortening, ejection fraction, and LVSP in dSG KO mice, and decreased plasma ANP. However, it had no effect on the contraction deficits of isolated cardiomyocytes other than to normalize the slowed relaxation of dSG KO. Ifetroban-treated mice had improved myocardial, but not skeletal muscle fibrosis. This was most noticeable in the LV free wall and occurred in conjunction with decreased TGF-beta activity and normalized plasma WBC. Typical of DMD cardiomyopathy, dSG KO hearts had reduced expression of neuronal nitric oxide synthase (nNOS) and claudin-5, which was improved with TPr antagonism. The results of our studies indicate that TP receptor activation may contribute to MD cardiomyopathy, and oral antagonism of the TP receptor may be a novel therapeutic for the cardiac phenotype in DMD and LGMD patients.
Abstract 398: GLP-1 Receptor Agonist Administered After the Infarct Suppresses miR-33 Expression in Lean Mice Subjected to Permanent Coronary Artery Occlusion
Authors: de F. Germano Juliana , Huang Chengqun , Song Yang , Sin Jon , Mentzer Robert M , A. Gottlieb Roberta , Andres Allen M
Published: February 2019

MicroRNAs (miRs) regulate post-transcriptional gene expression and protein translation. There is now increasing evidence that miRs regulate mitochondrial turnover. miR-33 regulates cholesterol metabolism and mitochondrial function in cardiac fibroblasts and macrophages and has been implicated in promoting fibrosis. Excessive fibrosis contributes to the development of adverse LV remodeling. Given our recent findings that a glucagon-like peptide-1 receptor agonist (GLP1Ra) [Sigma] induces mitophagy and mitigates post-MI LV remodeling, the purpose of this study was to determine its effects on miR-33 expression after MI. We analyzed miR-33 expression in the viable regions of hearts in lean mice after permanent coronary arterial ligation (PCAL) and subsequent treatment with GLP1Ra administered 2h and 48h after the infarction (total of 2 doses). PCAL was associated with a marked increase in miR-33 that peaked at day 3 (data not shown). In the presence of GLP1Ra, miR-33 expression was suppressed 3 days post-PCAL (
A
). Polysome profiling revealed that miR-33 was decreased in the translating fractions of GLP1Ra-treated mice and increased in the non-translating fraction (80S free ribosomes) compared to vehicle (
B
and
C
). Decreased association of miR-33 with polysomes in GLP1Ra-treated mice would allow for increased target translation. Given that suppression of miR-33 is implicated in promoting fibrosis, a suppression of this miR by acute GLP1Ra treatment could explain the beneficial effects of this drug in limiting adverse cardiac remodeling.
Abstract 399: Inhibition of NFE2L2-ARE Pathway by mtROS Contributes to Development of Cardiomyopathy and Left Ventricular Dysfunction in Chagas Disease
Authors: Wen Jake J , Garg Nisha J
Published: February 2019

Aims:
We investigated the effects of mitochondrial reactive oxygen species (mtROS) on nuclear factor (erythroid 2)-like 2 (NFE2L2) transcription factor activity during Trypanosoma
cruzi
(Tc) infection and determined whether enhancing the mtROS scavenging capacity preserved the heart function in Chagas disease.

Results:
C57BL/6 wild type (WT, female) mice infected with Tc exhibited myocardial loss of mitochondrial membrane potential, complex II (CII)-driven coupled respiration, and ninefold increase in mtROS production. In vitro and in vivo studies showed that Tc infection resulted in an ROS-dependent decline in the expression, nuclear translocation, antioxidant response element (ARE) binding, and activity of NFE2L2, and 35-99% decline in antioxidants’ (gamma-glutamyl cysteine synthase [γGCS], heme oxygenase-1 [HO1], glutamate-cysteine ligase modifier subunit [GCLM], thioredoxin (Trx), glutathione S transferase [GST], and NAD(P)H dehydrogenase, quinone 1 [NQO1]) expression. An increase in myocardial and mitochondrial oxidative adducts, myocardial interventricular septum thickness, and left ventricle (LV) mass, a decline in LV posterior wall thickness, and disproportionate synthesis of collagens (COLI/COLIII), αSMA, and SM22α were noted in WT.Tc mice. Overexpression of manganese superoxide dismutase (MnSOD) in cultured cells (HeLa or cardiomyocytes) and MnSODtg mice preserved the NFE2L2 transcriptional activity and antioxidant/oxidant balance, and cardiac oxidative and fibrotic pathology were significantly decreased in MnSODtg.Tc mice. Importantly, echocardiography finding of a decline in LV systolic (stroke volume, cardiac output, ejection fraction) and diastolic (early/late peak filling ratio, myocardial performance index) function in WT.Tc mice was abolished in MnSODtg.Tc mice.

Innovation and Conclusion:
The mtROS inhibition of NFE2L2/ARE pathway constitutes a key mechanism in signaling the fibrotic gene expression and evolution of chronic cardiomyopathy. Preserving the NFE2L2 activity arrested the mitochondrial and cardiac oxidative stress, cardiac fibrosis, and heart failure in Chagas disease.
Abstract 408: Defective Mitochondrial Dynamics Contribute to Cardiac Contractile Dysfunction in Desminopathy
Authors: Alam Shafiul , Abdullah Chowdhury S , Aishwarya Richa , Miriyala Sumitra , Panchatcharam Manikandan , Green Jonette P , Orr A. W , James Jeanne , Robbins Jeffrey , Bhuiyan Md. S
Published: February 2019

Rational:
Desmin plays a critical role in the maintenance of the structural and mechanical integrity of the sarcomere. Mutation in desmin causes disorganization of contractile apparatus leading to cardiomyopathy. Cardiomyocyte-specific expression of mutated desmin (a seven amino acid deletion R172-E178, D7-Des Tg) causes accumulations of toxic protein aggregates leading to cardiac contractile dysfunction. Whether these alterations result in maladaptive changes in mitochondrial dynamics and contribute to mitochondrial respiratory defects resulting in cardiomyopathies remains obscure.

Objective:
In this study, we investigated whether altered mitochondrial dynamics play a causative role in the development of cardiac contractile dysfunction and cardiomyocyte death in D7-Des Tg mouse model of desminopathy.

Methods and results:
D7-Des Tg hearts showed accumulation of desmin aggregates resulting in disruption of the desmin network, development of cardiomyocyte hypertrophy, and contractile dysfunction. Ultrastructural analysis by electron microscopy showed highly perturbed mitochondrial spatial organization and mitochondrial fragmentation in D7-Des Tg mice. D7-Des Tg mice hearts showed increased mtDNA copy number, increased expression of mitochondrial fission regulatory proteins, defects in mitochondrial respiration and activation of apoptotic cell death. Adenoviral-mediated expression of D7-Des in cardiomyocytes also showed increased mitochondrial fission, expression dependent inhibition of mitochondrial respiration and activation of cellular toxicity. Inhibition of mitochondrial fission by mitochondrial division inhibitor mdivi-1 significantly improved mitochondrial respiration and inhibited cellular toxicity associated with D7-Des expression in cardiomyocytes.

Conclusion:
Aberrant mitochondrial fission results in mitochondrial respiratory defects and apoptotic cell death in a D7-Des Tg hearts. Inhibition of aberrant mitochondrial fission using mitochondrial division inhibitor significantly preserved mitochondrial function and decreased apoptotic cell death. Therefore, our study shows that maladaptive aberrant mitochondrial fission causes desminopathy-associated cellular dysfunction.
Abstract 415: Lin28a Regulates Pathological Cardiac Hypertrophic Growth Through Pck2-mediated Enhancement of Anabolic Synthesis
Authors: Ma Hong , Yu Shuo , Liu Xiaojing , Zhang Yingao , Fakadej Thomas , Liu Ziqing , Yin Chaoying , Locasale Jason W , Taylor Joan M , Qian Li , Liu Jiandong
Published: February 2019

During pathological hypertrophy, the heart undergoes extensive metabolic changes prior to the onset of structural remodeling, yet, it remains unknown whether and how this metabolic remodeling could contribute to cardiac hypertrophic growth. Here, we identified the RNA-binding protein Lin28a as a critical regulator of pathological cardiac hypertrophy and metabolic re-patterning. Cardiac specific deletion of
Lin28a
attenuated pressure overload-induced hypertrophy and cardiac dysfunction. Transcriptomics and metabolomic analysis highlighted roles for Lin28a in promoting cardiac glycolysis and anabolic biosynthesis. Mechanistically, Lin28a directly bound to mitochondrial phosphoenolpyruvate carboxykinase 2 (
Pck2
) mRNA and positively modulated its transcript level. By utilizing the neonatal rat cardiomyocytes (NRCMs) hypertrophy model, we found that manipulation of
Pck2
expression phenocopied the metabolic and hypertrophic phenotypes of manipulating
Lin28a
expression. Furthermore, epistatic analyses demonstrated that overexpression of
Pck2
reversed the attenuation of norepinephrine-induced enhancement of cardiac glycolysis and cardiac hypertrophy by loss of
Lin28a
function, and knockdown of
Pck2
suppressed Lin28a-induced increase in cardiac glycolysis and cardiomyocyte hypertrophic growth. Thus, our study revealed a critical role of Lin28a in the regulation of pathological cardiac hypertrophic growth through Pck2-mediated regulation of cardiac metabolism.
Abstract 425: A Pro-reductive Redox State Protects the Myocardium From Isoproterenol-Induced Pathological Remodeling in Nrf2 Transgenic Mouse
Authors: Shanmugam Gobinath , Challa Anil Kumar , Litovsky Silvio H , Fernandes Jolyn , Jones Dean P , Namakkal-Soorappan Rajasekaran
Published: February 2019

Background:
Nuclear factor (erythroid-derived 2)-like 2 (NEFL2/Nrf2) is an inducible transcription factor that is crucial to fight against stress caused by various means (i.e. oxidative or xenobiotic stresses). While the Nrf2 has a negligible role on regulating the basal transcription of antioxidant and cytoprotective genes, its activation in response to stress is indispensable. Here, we hypothesize that augmentation of myocardial Nrf2, through transgene expression, will protect the myocardium from isoproterenol-induced pathological remodeling.

Methods:
Cardiac specific Nrf2 transgenic (Nrf2-Tg) and non-transgenic (NTg) litter mates in the C57/BL6 background were subjected to isoproterenol (ISO) treatment (50 mg/kg bw/day, for 7 days), and assessed the myocardial structure, function (Echocardiography) and Nrf2-dependent defense mechanisms.

Results:
Severe infarction and fibrosis along with increased inflammation leading to myocardial dysfunction in NTg mice exposed to ISO, but the Nrf2-TG hearts were found to be resistant to ISO insult. Suppression of infarction and protection of myocardial structure and function in the Nrf2-Tg mice could be attributed to a pro-reductive condition displayed in these hearts. In particular, ISO administration resulted in an excessive generation of reactive oxygen species and oxidation of proteins along with significant downregulation of antioxidant genes (p<0.05)and their proteins lead to myocardial damage in NTg mice. Of note, myocardium of ISO-treated TG mice displayed significantly reduced protein oxidation and comparable levels of antioxidant genes and proteins (p<0.05) in relation to PBS-treated NTg mice. Taken together, it is suggested that a pre-existing pro-reductive redox milieu is likely to combat ISO-induced oxidative stress, thereby protecting the myocardium from pathological remodeling.

Conclusion:
Thus, we conclude that a modest trans-activation of the global antioxidant defense system might have therapeutic potential than either targeting single antioxidant (i.e. SOD or catalase or GPX) or supplementation of antioxidant combos to protect the myocardium in response to stress.
Abstract 427: Inhibition of Myocardial Romk Channels Blocks Ischemic Preconditioning Induced Cardio-protection
Authors: Ramratnam Mohun , Linnerooth Warren , Kyle John W , Weidmeyer Brandi A , Lacount Emily D , Ahmad Nihal , Pasternak Alexander , McNally Elizabeth M , Makielski Jonathan C
Published: February 2019

Background:
Prior evidence has shown that the renal outer medullary potassium channel (Kir 1.1 or ROMK) may be the pore forming subunit of the mitochondrial KATP channel (mitoKATP). However, it is unknown if ROMK inhibition affects myocardial function after ischemia reperfusion injury or cardio-protection from ischemic preconditioning.

Methods:
C57/BL6J mice were subjected to
ex vivo
ischemia (45 min) and then reperfusion (60 min) with or without ischemic preconditioning (3 cycles of 3 min). Mice were treated throughout the protocol with either 5 μM of compound A, a potent ROMK inhibitor, or vehicle, in a modified Krebs Henseliet buffer.

Results:
WT mice hearts subjected to ischemia reperfusion injury and perfused with 5 μM compound A recovered 73% of baseline left ventricular developed pressure (LVDP) while mouse hearts perfused with vehicle recovered 50% of baseline LVDP (P=0.07, N=3). Infarct size was not significantly different between hearts perfused with 5 μM compound A or vehicle (32% ± 7% vs. 46% ± 10%,
P
=0.18, N=3). In vehicle perfused mice, IPC significantly improved % recovery of LVDP (IPC 87% ± 9% vs. no IPC 50% ± 7%,
P
=0.014, N=3) and infarct size (IPC 22% ± 4% vs no IPC 46% ± 11%,
P
=0.02, N=3). However, IPC did not improve % recovery of LVDP (IPC 69% ± 6% vs no IPC 73% ± 4%,
P
=0.33, N=3) or infarct size (IPC 46% ± 14% vs no IPC 32% ± 7%,
P
=0.22, N=3) in mouse hearts perfused with compound A.

Conclusions:
Myocardial ROMK inhibition does not increase IR injury in an
ex vivo
mouse preparation, but does block cardio-protection from IPC further supporting ROMK as the potential pore forming channel of mitoKATP.
Abstract 429: Intensive Necroptosis Due to Enhanced RIP3-CaMKII Signaling Contributes to Diabetic Myocardial Ischemic Susceptibility
Authors: Xue Han , Li Chen , Yang Zheng , Wang Yishi , Mu Nan , Ma Heng
Published: February 2019

Diabetes increases the vulnerability of the heart to ischemic injury. Recent advances have shown that necroptosis is crucially involved in myocardial ischemia/reperfusion (I/R) injury. Our objectives were thus to extend our knowledge to whether and how necroptosis renders diabetic hearts more vulnerable to I/R injury, which involves activation of Ca
2+
-calmodulin-dependent protein kinase (CaMKII) by receptor-interacting protein 3 (RIP3). In this study, diabetes was induced by streptozotocin (40mg/kg, i.v.) and after 6 weeks, anesthetized open-chest diabetic and age-matched control C57 mice were subjected to 30-min regional ischemia (occlusion of LAD coronary artery) followed by 4-h reperfusion. Diabetic animals manifested significantly greater cardiac injury than their littermate controls. Notably, diabetic hearts were sensitized to I/R-induced myocardial necrosis, as evidenced by rise in lactate dehydrogenase (LDH) release and Evans blue dye (EBD) penetration. Notably, compared with control mice, the activation pattern of CaMKII in diabetic hearts subjected to I/R is altered by earlier advanced (60 min after reperfusion) phosphorylation and enhanced oxidation (21.5% increase). Then we proved that inhibition of CaMKII in early stage of I/R could effectively reduce myocardial necroptosis. Further, RIP3 expression and RIP3-CaMKII interaction are markedly enhanced in diabetic hearts during I/R. Inhibition of RIP3 markedly suppresses CaMKII activation and reduces necroptosis. Collectively, these findings identify enhanced RIP3-CaMKII pathway as a key regulator that increases myocardial necroptosis in diabetic I/R hearts.
Abstract 439: Elevating 2-deoxy Adenosine Triphosphate Improves Contraction in a Mouse Model of Genetic Dilated Cardiomyopathy
Authors: Moussavi-Harami Farid , Razumova Maria V , Klaiman Jordan M , Cheng Yuanhua , Tardiff Jil C , Regnier Michael
Published: February 2019

Several studies have identified reduced tension and Ca
2+
sensitivity as key regulators that precipitate dilated cardiomyopathy (DCM). We are developing a novel therapy to increase tension using adeno-associated viral (AAV) vectors that increase intracellular 2 deoxy-ATP (dATP) via overexpression of the enzyme ribonucleotide reductase (RNR). We have reported that dATP substitution for ATP increases the magnitude and rate of contraction and that RNR overexpression increases intracellular dATP and enhances cardiac function.

Here we use a multiscale approach to test the effect of dATP in a genetic model of dilated cardiomyopathy (DCM) with a missense mutation (D230N) in alpha-tropomyosin (Tm). Using recombinant proteins in the
in vitro
motility assay, we found that filament velocity was lower with D230N compared to WT Tm (2.03±0.04 vs. 2.44±0.04 μm/s). This deficit was corrected when dATP was substituted for ATP (2.90±0.04 μm/s). Next, we used transgenic D230N mice that show progressive ventricular dilation and systolic dysfunction. Intact and demembranated trabeculae from these mice have decreased tension and calcium sensitivity of force, and loss of length-dependent activation (LDA) compared to WT mice. Substitution of dATP for ATP, increased Ca
2+
sensitivity in D230N Tm myocardium (pCa
50
=5.48±0.02 vs. 5.57±0.02) and restored LDA. Complete replacement of ATP with dATP resulted in an ~70% increase in force at submaximal [Ca
2+
] and force at submaximal Ca
2+
was significantly increased by replacement of only 5% of the nucleotide pool with dATP. For isolated myofibrils from D230N Tm and WT mice, tension at submaximal Ca
2+
(pCa=5.6) was lower in D230N mice compared to WT mice (51±4 vs. 75±8 mN/mm
2
) and this deficit was improved with dATP (68±5 mN/mm
2
). Similarly, the rate of activation in D230N myofibrils was slower than in WT mice (2.1±0.2 vs.3.3±0.2 s
-1
) and partially corrected with dATP (2.7±0.2 s
-1
). These results demonstrate that enhanced cross bridge binding, via increasing dATP, can recover lost contractile function in a thin filament mutation model of DCM. In ongoing studies, we are testing the ability of elevated dATP, via AAV- RNR, to protect against reduced systolic function and progressive ventricular dilation in 2-3 week old D230N mice
.
Abstract 444: Dusp4 Overexpressed EC Derived Exosomes Promote Angiogenesis: A Novel Therapeutic Strategy For Ischemic Heart Disease
Authors: Dougherty Julie , Khan Mahmood , Angelos Mark G , Chen Chun-an
Published: February 2019

After an ischemia and reperfusion (I/R) injury to the heart, re-establishing blood flow to the infarct region can salvage myocardium, stimulate angiogenesis and recruit progenitor cells for tissue regeneration. Stimulating angiogenesis through the growth of new microcirculation in the peri-infarct area can help preserve and improve cardiac function following myocardial infarction. In this study, we hypothesize that exosomes derived from DUSP4-overexpressed endothelial cells (ECs) stimulate angiogenesis and improve vascular function. DUSP4-derived exosomes (50 μg/ml) promote angiogenesis by increasing tube formation of endothelial cells (2.74 ± 0.14 versus control exosomes 2.17 ± 0.15 in the total length; p < 0.001). Immunoblotting reveals that DUSP4 overexpression in endothelial cells down-regulates p53 (0.59 ± 0.04 versus 1.0 ± 0.01 of control) expression, which modulates the HIF1α pathway and angiogenesis. Moreover, NO is a critical small molecule involved in regulation of vascular tone, vascular growth, platelet aggregation, and modulation of inflammation. The level of NO generation determined using DAF fluorescence microscopy demonstrated that DUSP4 overexpression augments NO generation. The increase in NO generation also correlates to the increase in eNOS protein (1.23 ± 0.20 versus control) and mRNA (5.84 ± 0.37 versus control) expression. The proteomic analysis of control and DUSP4 derived exosomes showed that DUSP4 overexpression up-regulates several key exosomal proteins including HSP70, HSP90, tubulins, Vimentin, and clathrin. More interestingly, several redox proteins were up-regulated in DUSP4 derived exosomes including glutathione S-transferase P, peroxiredoxin-2, peroxiredoxin-4, and protein disulfide-isomerase. Together, these results demonstrate that exosomes derived from DUSP4 reprogrammed ECs are a valuable therapeutic option for the treatment of ischemic heart disease by promoting angiogenesis.
Abstract 445: Essential Role of the S100A1-C-terminus Domain in Maintaining Cardiac-inotrope S100A1 Protein Stability
Authors: Jebessa Zegeye H , Zhao Jemmy , Schneider Andrea , Busch Martin , Katus Hugo A , Most Patrick
Published: February 2019

Introduction:
S100A1 is a type of S100 protein with two EF-hand motif. It has a unique characteristic of interacting with multiple proteins of contractile machinery & mitochondria. S100A1 protein able to govern proper systolic & diastolic performance of cardiomyocyte interacting, respectively, with SERCA2A & RYR2. S100A1 level decreases dramatically as a consequence of cellular cues such as oxidative-stress or activation of cellular proteasomes. Selective reconstitution of reduced S100A1 level by AAV9-gene transfer rescued failing heart. Because of the unique ability of S100A1 to interact with myriads of proteins with multiple function, we aimed to dissect the structure-function relationship of the domains of S100A1.

Methods & results:
The C-terminus domain of S100A1 (S100A1ct) equally recapitulated the inotropic & other benefits of the S100A1-FL in experimental model of heart failure & identified as the bioactive lead domain of S100A1. To elucidate the function of the S100A1 without the C-terminus, here after, referred as S100A1ΔCT, we adenoviraly expressed S100A1ΔCT, & as a control, either expressing GFP or S100A1-FL. We, inadvertently, discovered that S100A1ΔCT could not be detected at protein level. PCR analysis & sequencing proved the presence S100A1ΔCT mRNA transcript. Bioinformatics revealed the presence of SUMO interaction motif (SIM) with in the C-terminus & multiple ubiquitination residues on S100A1ΔCT, suggesting the SIM domain of the S100A1 is crucial for stability. Inhibition of cellular proteasome lead to the detection of the S100A1ΔCT protein in cells over expressing S100A1ΔCT. We overexpressed S100a1ΔCT, deletion mutants having part of the SIM motif (S100a1-SIM
-
), the entire motif (S100a1-SIM
+
) & the S100a1-FL. Whereas the overexpression of S100A1-SIM
+
& S100A1-FL were detected, S100A1ΔCT & S100A1-SIM
-
could not be detected at protein level.

Conclusion:
The C-terminus of S100A1 is essential for stability of S100A1, besides, its potent inotropic function. Taken together, we deciphered the previously unrecognized posttranslational modification governed S100A1 regulation, thus providing new insight into the development of novel therapeutic strategies for heart failure via posttranslational manipulation of S100A1.
Abstract 447: The Role of Extracellular cAMP in the Pathogenesis of Pulmonary Arterial Hypertension
Authors: Sassi Yassine , bueno-Beti Carlos , Jones Carly , Bonnet Guillaume , Hadri Lahouaria , Hajjar Roger J
Published: February 2019

Pulmonary arterial hypertension (PAH) is an uncommon, progressive, life-threatening and often fatal disease. Despite advances in PAH therapy, there is no cure for PAH, and new therapies need to be developed. Activation of G-protein-coupled receptors stimulates adenylyl cyclase, leading in vascular smooth muscle cells to an increase in intracellular cyclic adenosine monophosphate (cAMP) formation and reduced cell proliferation
in vitro
and
in vivo
. Yet, the last two decades of research have shown that there is more to the role of cAMP than ever expected from this molecule. Stimulated cells transport cAMP outside the cells, a process that is mediated by the multidrug resistance-associated proteins (MRPs). Herein, we investigated the role of extracellular cAMP in the lung and asked whether it may act on pulmonary vascular remodeling. By employing a fluorescence resonance energy transfer (FRET)-based sensor, we found that extracellular cAMP activates intracellular cAMP formation in human pulmonary artery smooth muscle cells (hPASMC) and endothelial cells (hPAEC). Extracellular cAMP, via binding of its metabolite adenosine to the type A2 receptor, reduced hPASMC and hPAEC proliferation and migration by controlling the PKA/CREB pathway. To test for a role of extracellular cAMP in the pulmonary vasculature, we used rat-monocrotaline and mouse-hypoxia as
in vivo
models of PAH. Rats treated with monocrotaline developed PAH with increased pulmonary artery pressures (PAP) and right ventricular (RV) hypertrophy. Extracellular cAMP infusion significantly prevented and reversed these structural changes. Extracellular-cAMP-treated rats displayed lower RV systolic pressure and Fulton index, as well as decreased PAP. In line with these observations, we found infused cAMP to potently repress RV hypertrophy, RV systolic pressure, perivascular lung fibrosis and pulmonary arteries remodeling in mice exposed to Sugen5416 and chronic hypoxia. Together, our results assign extracellular cAMP a potent regulatory role in pulmonary vascular cells, and suggest targeting the extracellular cAMP signaling pathway to limit pulmonary vascular remodeling and PAH.
Abstract 451: Impact of Resistant Starch Diet on Atherosclerosis in the Presence of Chronic Kidney Disease
Authors: Karaduta Oleg , Zybaylov Boris
Published: February 2019

Chronic kidney disease (CKD) affects approximately 10% (1 of 10) of the worldwide population and is associated with a significantly increased risk of developing cardiovascular disease (CVD) due to the acceleration of atherosclerosis. In particular, a high incidence of atherosclerosis occurs in chronic renal failure patients with uremia, but the pathogenesis of the events that contribute to uremic atherosclerosis is poorly understood. Inflammation, vascular stiffness likely resulting from endothelial dysfunction, and accelerated atherosclerosis are considered prominent factors contributing to the high rates of CVD in CKD.

Our lab aims to demonstrate structural and functional protection against atherosclerosis by resistant starch diet, will provide a rationale to evaluate its use in CKD patients.

At the moment, our preliminary result shows significant increase in the bacteroidetes-to-firmicutes ratio. In addition, we observed an increase in the Ruminoccoccus genus upon RS supplementation. Next, we applied our metaproteomics analysis pipe-line to assess proteome changes between the four groups - healthy, healthy+RS, CKD, and CKD+RS. Our data shows that RS diet stimulates bacterial mass biosynthesis (characterized by highly abundant transcription/translation-related proteins) and increases bacterial proteins that may participate in host urea cycle.

An understanding of the link between microbiota and development of atherosclerosis in presence of end stage renal failure is of considerable clinical relevance. Our further plans include implementing deeper statistical analysis to establish strong correlations between those changes and CKD/CVD outcome, as well as identifying a target agent for CVD treatment. In summary, our methodology results in data on both host and bacterial proteins and yields both peptide-level and protein-level information. Our studies provide a foundation for identification of peptide-level, protein-level, and organism-level biomarkers of advanced kidney disease and suggest novel therapeutic targets. There is significant potential for drugging the microbiome with dietary recommendations.
Abstract 454: Role of Foxf1 During Protein Quality Control in Cardiomyocytes
Authors: Bhandary Bidur , Meng Qing Hang , Xu Na , Osinska Hanna , Shay-Winkler Kritton , Gulick James , Robbins Jeffrey
Published: February 2019

Introduction:
Impairment of protein quality control leads to the accumulation of intracellular misfolded protein aggregates, contributing to cardiac disease and heart failure. Essential protein quality control pathways, including proteasomal degradation and autophagy, are involved in maintaining cellular homeostasis and cardiac function during proteotoxic insult. In a prior study, we undertook an unbiased, total genomewide screen for RNA transcripts and their protein products that affect aggregate accumulations in the cardiomyocytes. Forkhead box F1 (Foxf1) gene was one of the hits from our high throughput screening. In this study, we have performed further validation of Foxf1, a transcription factor and previously unknown player in proteotoxic processes.

Aims and Methods:
The objective of this study was to validate Foxf1 as a contributor to protein quality control and understand its role in contributing to protein aggregate formation during proteotoxic-induced cardiac disease. We knocked down Foxf1 expression using siRNA mediated transfection in neonatal rat ventricular cardiomyocytes that expressed the protein aggregate inducing mutation of the chaperone α B crystallin (CryABR120G). Proteasomal function and relative activity in cells was analyzed using a degron-destablized green fluorescent protein (GFPu) based reporter protein. Similarly, autophagy activity was determined by autophagic flux assay using bafilomycin treatment.

Results:
CryABR120G expression leads to aggregate formation and decreased proteasomal function in cardiomyocytes. Knockdown of Foxf1 gene expression in CryABR120G-transfected cardiomyocytes reduced the proteotoxic sequelae, decreasing aggregate concentration. Knockdown of Foxf1 significantly increased proteasomal function in the model without altering autophagy activity.

Conclusion:
Decreased Foxf1 expression reduced CryABR120G induced aggregate protein accumulation and decreased proteotoxic stress in cardiomyocytes, likely through the maintenance of clearance of the misfolded protein via the proteasomal pathway.
Abstract 458: Role of Heterochromatin Protein 1 Binding Partner 3, HP1BP3, in Cardiomyocyte Stress Response
Authors: Chan Cheryl X , Tan Wilson , Foo Roger
Published: February 2019

Cardiovascular disease (CVD) is a top cause of mortality and a serious economical burden. While many diseases fall under the umbrella of CVD, they all ultimately lead to heart failure. Little is known how and what epigenetic factors could be involved in cardiomyocytes stress response during such end stage event. The objective of this study is to uncover novel epigenetic proteins that might be nodule regulators of stress genes, to better understand and alter inducted stress pathways. Here, we found Heterochromatin Protein 1 Binding Partner 3,
Hp1bp3
, in our single nuclear transcriptomics data set of sham and TAC murine cardiomyocytes.
Hp1bp3
was up-regulated in a subset of TAC nuclei (53.8% in TAC versus 18.9% in sham) and appeared to be central in a module of disease-related cardiac genes, suggesting its possible role of stress response regulation. Indeed, a subset of DE genes seemed to be co-regulated with
Hp1bp3
only in TAC nuclei amongst other subsets of genes that are not co-regulated. In contrast, stress gene regulation in sham nuclei seemed to not be controlled by
Hp1bp3
. These data suggest that
Hp1bp3
is unique in diseased nuclei and could be an important regulator of specific stress genes. When we performed the Ingenuity Pathway Analysis, we observed that
Hp1bp3
-high TAC nuclei activated many cardioprotective pathways, like hypoxia-handling and ErBB-signaling pathways, suggesting
Hp1bp3
was up-regulated to initiate a cardioprotective stress-handling response. Further experiments with a transgenic HL-1 cell line over-expressing
Hp1bp3
showed a robust down-regulation of stress genes (Nppb, Myh7) after exposure to hypoxia.
Abstract 493: Serum Leptin and TNFa Levels in Relation to Systolic and Diastolic Heart Function in Obese and Normal-overweight Pregnant. First Trimester Data
Authors: Shreyder Katherine , Carrillo Maira , Maher James , Schlabritz-Lutsevich Natalia
Published: February 2019

Introduction:
Maternal obesity (MO) defined as pre-pregnancy body-mass index (BMI) >30kg/m2, is the strongest risk factors for preeclampsia (PE). Information regarding cardiac changes in MO as well as the role of inflammatory biomarkers in PE pathogenesis is conflicting.

The aim of this study
was to evaluate echocardiography changes in normal/ overweight pregnant (NP) (BMI 18.0-30) and obese pregnant women (OP) (BMI>30) and to correlate them to dynamics of biomarkers (leptin, TNFa, BNP, pentraxin-3, IL6) and mRNA.

Material and methods:
There were 11 OP (BMI 33.6±2.9) and 13 NP (BMI 25.5±2.3) enrolled into this study in the first trimester of pregnancy. All patients were nulliparous. There were no differences between OP and NP according to age and number of previous pregnancies. Patients with pre-pregnancy cardiovascular pathology were excluded. The 2D echocardiography with Doppler was performed in each pregnancy trimester. Echocardiography results, leptin and TNFa in the first trimester are available for now.

Results:
Left ventricular (LV) mass was significantly higher while ejection fraction (EF) and ratio of the early (E) to late (A) ventricular filling velocities (E/A ratio) appeared to be lower in OP, compared to NP (Table). Leptin was higher in OP. TNFa inversely correlated with stroke volume (SV), MO positively associated with leptin levels and negatively with E/A ratio (p<0.05).

Conclusion:
Differences in systolic and diastolic heart function between OP and NW are being evident already in the first trimester. Involvement of leptin and TNFa; might be suggested. Identifying biochemical mechanisms driving cardiac changes in MO are crucial for PE prevention.
Abstract 495: The Loss Of Pka Activity And Activation In Cardiomyocytes Does Not Induce Cardiac Abnormalities
Authors: Zhang Xiaoying , Zhang Ying , Li Ying , Ai Xiaojie , Zhang Xiaoxiao , Zhang Chen , Tang Mingxin , Xiang Hua , Houser Steven , Chen Xiongwen
Published: February 2019

Protein Kinase A (PKA) is the major mediator of β-adrenergic (βAR) regulation of cardiac contractility but other mediators (e.g., EPAC) have also been suggested. Reduced PKA basal activity and activation are linked to cardiac diseases. However, the roles of PKA in cardiac physiology and pathology have not been clearly defined because it has been difficult to knockout all expressed PKA catalytic subunit genes in cardiomyocytes. We ablated PKA activity and activation in cardiomyocytes by expressing a PKA inhibitor peptide (PKAi-GFP) in cardiomyocytes in transgenic (TG) mice. We made novel discoveries that: (1) Basal PKA-mediated phosphorylation was reduced for proteins in myofilaments and longitudinal SR but not for proteins in subsarcolemmal and dyadic spaces in PKAi TG hearts, suggesting basal PKA activity is different in individual cellular compartments; (2) PKAi increased the expression of CaMKII, the L-type Ca
2+
channel β subunits and current, but decreased CaMKII phosphorylation; (3) These changes resulted in significantly enhanced myofilament Ca
2+
sensitivity with prolonged contraction and relaxation, decreased myocyte Ca
2+
transient decay rate but enhanced myocyte contractility. (4) PKA inhibition fully abolishes the increases of heart rate, and cardiac and myocyte contractility by a β-adrenergic agonist (isoproterenol, ISO), showing a surprising dominant role of PKA in heart rate regulation and a minimal role of PKA-independent βAR signaling in regulating cardiac function; (5) The increased phosphorylation of proteins mediated by PKA and CaMKII after ISO was prevented by PKAi because increased PKA and CaMKII activation by ISO was eliminated by PKAi
in vivo
and
in vitro
. (6) PKAi TG mice maintained partial exercise capability by enhancing basal cardiac function, vascular constriction and ventricular filling (i.e., preload) during βAR stimulation. These features resemble some of the characteristics of diseased hearts. However, PKAi TG mice did not show any cardiac functional or structural abnormalities during the 1-year study period. In conclusion, PKA is the essential mediator of acute βAR regulation of cardiac function and exercise capacity, but the loss of PKA basal activity and activation by βAR agonists do not cause cardiac disease.
Abstract 498: Functional Annotation of
TNNT2
Variants of Uncertain Significance With Induced Pluripotent Stem Cell-derived Cardiomyocytes
Authors: Lv Wenjian , Qiao Lyon , Petrenko Nataliya , Li Wenjun , Owens Anjali T , McDermott-Roe Chris , Musunuru Kiran
Published: February 2019

The introduction of clinical sequencing is dramatically increasing the discovery of variants of uncertain significance (VUSs) in genes linked to inherited cardiomyopathies. We have established a platform for rapid and efficient insertion of
TNNT2
gene variants into an induced pluripotent stem cell (iPSC) line to generate an allelic series of isogenic clones for differentiation into cardiomyocytes (iPSC-CMs) for functional annotation of the variants. We first used CRISPR-Cas9 to introduce known pathogenic variants into iPSCs from a healthy person or to correct pathogenic variants in iPSCs from patients with severe cardiomyopathy. Whereas normal/corrected iPSC-CMs responded to isoproterenol treatment with a 50%-70% increase in spontaneous beating rate as assessed by patch-clamp studies, iPSC-CMs with pathogenic variants had minimal responses (close to 0%). Due to the inefficiency of CRISPR-Cas9 in introducing/correcting variants in iPSCs, we next used dual integrase cassette exchange (DICE) to allow for the introduction of a large number of variants in parallel into a pool of cells. In a single pilot use of the DICE platform, we isolated heterozygous clones with 14 unique variants, >10% of all
TNNT2
coding variants cataloged in ClinVar. We found that iPSC-CMs with any of 7 VUSs or likely pathogenic variants were impaired in their response to isoproterenol, in contrast to control DICE-treated iPSC-CMs. Finally, we sought to apply the DICE platform to a patient case in real time. A 65-year-old woman with severe hypertrophic cardiomyopathy underwent gene panel testing that identified a single VUS,
TNNT2
E251D. Between the first and second clinic visits (~10 weeks), we were able to use DICE to rapidly and efficiently generate iPSC-CMs with the E251D variant and determine they had normal responses to isoproterenol, suggesting that the variant is not pathogenic. Guided by this finding, we recommended that the patient’s children and grandchildren not undergo cascade genetic screening for the E251D variant. In conclusion, this work establishes the feasibility of rapid functional annotation of cardiomyopathy gene variants, which after further validation could be incorporated into clinical practice as a line of evidence to support variant classification.
Abstract 501: Anemia of Inflammation in an Exonuclease Deficient Lupus Mouse
Authors: Rego Stephen L , Harvey Scott , Simpson Sean R , Hemphill Wayne O , Grayson Jason M , Perrino Fred W
Published: February 2019

Background:
Patients with chronic inflammatory conditions, such as autoimmune diseases, infections, and cancer, often display anemia of inflammation (AI). 50% of patients with systemic lupus erythematosus display clinical signs of anemia. Mutations at aspartate residue 18 of the three prime repair exonuclease 1 (
TREX1
) gene, which codes for the primary mammalian exonuclease, cause a monogenic form of cutaneous lupus erythematosus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease.

Objective:
The link between failed DNA degradation during erythropoiesis and anemia prompted our studies to investigate whether TREX1 dysfunction might contribute to AI.

Methods:
AI in TREX1 D18N mice was determined through analysis of hematocrit, hemoglobin, gene expression analysis, immunohistochemistry (IHC) and flow cytometry. Response to phenylhydrazine (PHZ) induced anemia and activation of DNA sensing pathways were determined through IHC and interferon stimulatory gene (ISG) expression analysis of the liver, spleen and erythroblastic islands.

Results:
The TREX1 D18N mice exhibit altered erythropoiesis including reduced hematocrit, increased erythropoietic and hematotoxic gene expression levels in the spleen (~10-20 fold) and phenotypic signs of normocytic normochromic anemia. Anemia in TREX1 lupus mice is accompanied by increased erythropoietin (Epo) and extramedullary hematopoiesis. Furthermore, the TREX1 D18N mice display an inappropriate response to anemic challenge. Enhanced ISG expression is apparent and results from failed processing and sensing of undegraded erythroblast DNA.

Conclusion:
These results support a critical role for TREX1 in the degradation of erythroblast DNA and identify a distinct AI phenotype in TREX1 D18N mice.
Abstract 506: Modulation of ZIC3 Expression in Pluripotent Stem Cells Using CRISPR-interference to Model Heterotaxy
Authors: Liu Bohao , Lock Roberta , Vunjak-Novakovic Gordana , Fine Barry
Published: February 2019

Failure of proper left-right patterning during embryogenesis is an underlying mechanism of congenital heart disease (CHD). Heterotaxy (HTX) is a complex laterality syndrome in which multiple organs, including the heart, are abnormally arranged and mal-rotated. Loss-of-function mutations in
ZIC3
, a zinc finger transcription factor within the GLI superfamily, have been associated with X-linked HTX. The mechanism behind how ZIC3 causes laterality defects remains unknown. To investigate this relationship, we developed a method to directly explore ZIC3 using induced pluripotent stem cells (iPSCs) at a molecular, cellular, and functional level through utilization of CRISPR interference (CRISPRi). A stable CRISPRi transgenic line was generated through lentiviral transduced vectors encoding inducible endonuclease-dead Cas9 and an sgRNA targeting the ZIC3 promoter. As ZIC3 is necessary for iPSC pluripotency, this inducible system allowed for a controllable knockdown while maintaining parent cell line pluripotency. RT-PCR confirmed that doxycycline induced a robust knockdown of ZIC3 expression over 72 hours with a fold change of 0.12 compared to non-induced. RNA-Seq was used to generate a gene expression profile of ZIC3 loss. This showed that 446 genes were significantly differentially expressed (adjusted p < 0.01), and of those, 163 genes had an absolute fold change of greater than 1.5. Of the top 15 most significantly differentially expressed genes, 47% (7 of 15) are associated with the cytoskeleton, including gamma actin 1 and myosin light chain 9. These results were confirmed by RT-PCR. Confocal imaging revealed significant changes to the actin cytoskeleton in response to ZIC3 knockdown, with a notable reduction in filamentous actin. Current ongoing studies are focusing on the impact of ZIC3 knockdown on the structural and mechanical differences in differentiated cardiomyocytes. In summary, we have developed a method by which to explore CHD using iPSCs in order to better understand the genetic mechanisms of heterotaxy, and in future studies, other forms of CHD.
Abstract 526: Free Fatty Acid Receptor 4 is Required for an Adaptive Response to Pathologic Pressure Overload-induced Heart Failure in Mice
Authors: Joshi Sonal S , Healy Chastity L , Wagner Brandon M , O’Connell Timothy D
Published: February 2019

Background:
Our previous results indicate that eicosapentaenoic acid (EPA) prevents interstitial fibrosis and pathologic remodeling in the transverse aortic constriction (TAC) model of pressure overload induced heart failure in mice. However, EPA supplementation did not lead to incorporation into cardiac myocytes or fibroblasts
in vivo
, a traditional mechanism of action. Interestingly, we found that free fatty acid receptor 4 (Ffar4), a G-protein coupled receptor for long-chain fatty acids, was expressed in cardiac myocytes and fibroblasts. In primary cultures of cardiac fibroblasts, Ffar4 was sufficient and required to prevent TGFβ1-induced fibrosis, suggesting Ffar4 might mediate EPA cardioprotection.

Methods:
To understand the function of Ffar4 in the heart
in vivo
, we examined the response to TAC in male and female Ffar4 knockout mice (Ffar4KO) under basal conditions without EPA supplementation.

Results:
In male Ffar4KO mice, TAC induced a worsened dilated cardiomyopathy relative to wild-type (WT) mice. Specifically, in male Ffar4KO mice, TAC produced more hypertrophy (Heart weight: WT: 175±8 mg, n=14; Ffar4KO: 223±14 mg, n=10;
P
<0.05), and generated more severe systolic and diastolic dysfunction (Ejection Fraction: WT: 42±3%, n=14; Ffar4KO: 28±4%, n=10;
P
<0.05) (E/A ratio: WT: 2.0±0.2, n=12; Ffar4KO: 3.7±0.5, n=9;
P
<0.05). However, TAC induced similar levels of interstitial fibrosis in male Ffar4 KO and WT mice (WT: 9.6±1.8%, n=14; Ffar4KO: 7.4±1.1%, n=10;
ns
). While TAC worsened outcomes in male Ffar4KO mice, TAC in Ffar4 KO female mice produced no significant differences in the degree of hypertrophy or contractile dysfunction relative to WT mice.

Discussion:
Our results indicate that Ffar4 is required for an adaptive response to pathologic pressure overload in male mice. The lack of an exaggerated fibrotic response was surprising given our previous results indicating that in cardiac fibroblasts, Ffar4 prevents TGFβ1-induced fibrosis. This might indicate that Ffar4 signaling is minimal in fibroblasts under conditions of low omega-3 fatty acid levels. This result also suggests that the worsened contractile dysfunction in Ffar4KO mice is unrelated to fibrosis, but involves a direct effect in cardiac myocytes.