bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 148: Neonatal Mouse Heart Regeneration is Dependent on Mononuclear Diploid Cardiomyocytes and Paracrine IGF2 Signaling
Authors: Shen Hua , Patterson Michaela , Gan Peiheng , Sucov Henry M
Published: October 2019

In normal mice, injury to the newborn heart is efficiently regenerated, but this capacity is lost by one week after birth when most cardiomyocytes have become post-mitotic. Concurrently during the first week, most cardiomyocytes become polyploid. Despite this temporal association, the relation of polyploidization to neonatal heart regeneration has not been adequately examined. In this study, we find that IGF2, an important mitogenic factor in heart development, is required to support neonatal mouse heart regeneration. Following injury on postnatal day 1, absence of IGF2 abolished injury-induced cell cycle entry (as indicated by phospho-histone H3 staining) during the early part of the first postnatal week, when cardiomyocytes are still mononuclear and diploid. Consequently, regeneration failed despite the later presence of additional activities that support robust cell cycle entry 7 days following injury, a time when most cardiomyocytes are polyploid and no longer able to complete cytokinesis. IGF2 originates from the endocardium/endothelium lineage and is transduced by the insulin receptor; both features distinguish the action of IGF2 in neonatal heart regeneration from that in embryonic heart development. Regeneration in injured Igf2-deficient neonates was rescued by three different contexts that elevate the percentage of mononuclear diploid cardiomyocytes beyond postnatal day 7. Thus, IGF2 is the primary paracrine-acting mitogen for heart regeneration during the early postnatal period, and IGF2-deficiency unmasks the dependence of this process on proliferation-competent mononuclear diploid cardiomyocytes.
Abstract 152: Tip60 Depletion Promotes Cardiomyocyte Proliferation and Attenuates Ischemic Injury in the Adult Heart
Authors: Wang Xinrui , Wan Tina C , Lupton Carri , Harrison Mitchell , Lough John , Auchampach John A
Published: October 2019

The pathogenesis of myocardial infarction (MI) is largely attributed to the loss of cardiomyocytes (CMs) and their insufficient regeneration. Inducing the proliferation of pre-existing CMs has emerged as a potential therapeutic strategy for cardiac repair. Results in our laboratory indicate that Tip60 (Tat-interactive protein 60 kD), a pan-acetylase protein encoded by the
Kat5
gene, inhibits CM proliferation consequent to its induction of the DNA damage response (DDR) at neonatal stages, which has recently been shown to cause CM replicative senescence. To determine whether Tip60 depletion permits re-entry of adult CMs into the cell-cycle and confers protection from MI, we are employing a line of

Kat5
flox/flox

mice wherein Tip60 is conditionally and specifically depleted in CMs via tamoxifen-induced activation of a
Myh6
-driven merCremer transgene. In uninjured hearts, Tip60 depletion results in transient thickening of the left ventricular walls, accompanied by markedly increased expression of G
2
/M-phase cell cycle regulators (cyclins A2 & B1, Cdk1) and de-differentiation markers (Myh7, Osm, OsmR, Runx1), diminished CM size, decreased expression of cell-cycle inhibitors (p27, Meis1), and remarkable increases in Ki67 and pH3-positive CMs as well as non-CMs. In hearts infarcted by permanent ligation, tamoxifen administration increases fractional shortening, ejection fraction, and anterior wall thickening within 7 days, conditions that are sustained for at least 18 additional days, when reduced scarring is indicated by trichrome staining. Taken together, these results indicate that Tip60 depletion in adult heart may preserve cardiac performance after MI by inducing CM regeneration. These findings should help advance our understanding of the molecular mechanisms that keep CMs in replicative senescence, establishing a possible therapeutic target for maintaining and restoring cardiac muscle after MI.
Abstract 182: Left Ventricular Diastolic Dysfunction in HIV-infected ART-naive and HIV-negative Tanzanian Adults: A Cross-sectional Study
Authors: Kingery Justin , Damas Edmund , Kalluvya Samuel , Pirmohamed Altaf , Goyal Parag , Hee Lee Myung , Kalokola Fredrick , Desdarius Bernard , Peck Robert
Published: October 2019

Objective:
To compare the prevalence and associated factors of left ventricular diastolic dysfunction in HIV-infected, ART naïve and HIV-negative Tanzanian adults.

Methods:
A cross-sectional analysis of a longitudinal study including: 257 HIV-infected, ART naïve adults and 265 HIV-negative controls. Echocardiography and traditional risk factors were performed and determined by standard investigations. The primary outcome was prevalence of left ventricular diastolic dysfunction. Secondary outcomes were factors associated with and grade of dysfunction.

Results:
Compared to HIV-negative controls, HIV-infected, ART naïve adults had a 2-fold higher prevalence of diastolic dysfunction (OR=2.06 [1.16-3.66], p=0.01). Additionally, age, female sex, obesity and hypertension were significantly associated with dysfunction in HIV-negative adults; whereas, only age and hypertension were associated with dysfunction in HIV-infected adults. As compared to HIV-negative controls, significantly more HIV-infected, ART naïve adults had higher grade dysfunction (p=0.02) and more ventricular hypertrophy. HIV-infected adults with diastolic dysfunction also expressed higher levels of inflammatory cytokines including TNFa, IL6, IL8, IL33 and sST2 as compared to both HIV-negative and HIV-infected adults without dysfunction.

Conclusions:
HIV-infected Tanzanian adults have a 2-fold higher prevalence of left ventricular diastolic dysfunction in the period immediately following diagnosis and before ART initiation as compared to HIV-negative adults, and this dysfunction is of higher grade and is associated with myocardial hypertrophy. These data identify diastolic dysfunction in an international cohort in the immediate post-diagnosis, pre-ART period similar to that previously reported in the post-ART period in high-income countries. Additionally, traditional cardiovascular risk factors are not significantly associated with dysfunction in HIV-infected adults; however, cytokine patterns are present and may be important for risk stratification in this group. Therefore, studies delineating appropriate screening for this population are needed.
Abstract 186: Superiority of Sacubitril/Valsartan Over Valsartan Alone in Attenuating Pressure Overload-induced Cardiac Fibrosis and Oxidative Stress
Authors: Li Xiaofei , Braza Julie , Mende Ulrike , Zhang Peng
Published: October 2019

Background:
Sacubitril/valsartan, a first-in-class Angiotensin Receptor-Neprilysin Inhibitor (ARNi), is superior to valsartan in reducing blood pressure in hypertensive patients. However, cardiac effects of sacubitril/valsartan in hypertension treatment and its potential superiority over valsartan are unclear. This study was designed to investigate effects of sacubitril/valsartan on cardiac structural remodeling and oxidative stress using a pressure overload rat model induced by ascending aortic constriction (AAC) and to address whether direct regulation of cardiac myocytes and/or fibroblasts is involved in the cardiac effects independent of afterload changes.

Methods and Results:
1 week after AAC surgery, adult male Sprague-Dawley rats were randomized for 10 weeks treatment with vehicle, valsartan, or sacubitril/valsartan. Sacubitril/valsartan was superior to valsartan in inhibiting ventricular fibrosis, while both equally inhibited cardiomyocyte hypertrophy at the cellular level. These findings were supported by
in vitro
experiments showing that combined LBQ657 (sacubitrilat, active form of sacubitril) and valsartan was superior to valsartan alone in inhibiting angiotensin II (Ang II)-induced adult ventricular fibroblast activation, but not adult cardiomyocyte hypertrophy. In adult cardiomyocytes LBQ657 effectively inhibited Ang II-induced mitochondrial superoxide, and combined LBQ657 and valsartan was superior to valsartan or LBQ657 alone. Importantly, sacubitril/valsartan was also superior to valsartan in reducing mitochondrial superoxide in cardiomyocytes from pressure-overloaded hearts and improved cardiomyocyte mitochondrial maximal and spare respiration capacity.

Conclusions:
These findings provide direct evidence and novel mechanistic insights on the superior cardioprotective effects of sacubitril/valsartan in the setting of pressure overload: independent of afterload reduction, sacubitril/valsartan is superior to valsartan in attenuating cardiac fibrosis and myocyte oxidative stress and improves myocyte mitochondrial function in rats. Our study thus suggests a greater therapeutic effect of sacubitril/valsartan in the early stage of hypertensive heart disease before heart failure.
Abstract 192: Sex Dependent Differences in Cardiac Myocyte Excitation-Contraction Coupling After Chronic Stimulation of the Delta or Kappa Opioid Receptors During Normoxic and Hypoxic Conditions
Authors: Klos Matthew , Ahmad Ramiz , Pua Isabella , Hicks Kayla , Suresh Shreyas , Morgenstern Sherry , Devaney Eric
Published: October 2019

Introduction:
In 2016 the US government declared opioid misuse a public emergency. While most opioid related deaths can be attributed to overdose, recent data suggest a link between opioid use and cardiovascular disease. Further, epidemiological data suggest it may be sex dependent, with females (♀) being at a somewhat higher risk than males (♂). This is paradoxical because until menopause, ♀ have a lower risk of heart disease when compared to ♂. Healthy adult hearts express the δ and κ ORs, but not the μOR. Therefore, the overall goals of this study using a reductionist model were to: 1). To determine if there are sex specific differences in cardiac myocyte excitation-contraction coupling between ♂ and ♀ myocytes exposed to δ or κ OR agonists during normoxia/hypoxia. 2). Examine if 17β-Estradiol enhances or augments the effects of δ or κ OR stimulation.

Methods:
Myocytes were isolated from adult ♂ and ♀ Sprague-Dawley rats and cultured overnight in M199 HEPES media, or M199 HEPES media containing either 1 μM DADLE (δ agonist), 20 μM U50-488 (κ agonist), 100 nm 17β-Estradiol, 1μM DADLE + 100 nm 17β-Estradiol, or 20 μM U50-488 + 100 nm 17β-Estradiol. To simulate an infarct, a subset of the myocytes was incubated at 1%O
2
, 5% CO
2
for 4 hours + 10 nM isoproterenol followed by 60 min normoxia (21%O
2
, 5% CO
2
) before recordings. Excitation-Contraction was measured using an IonOptix rig (IonOptix LLC, Milton MA) at 36±1° at 1 Hz pacing.

Results:
During normoxia, ♂ myocytes compared to ♀ myocytes had an increased peak contraction and peak Ca
2+
transient. The addition of 100 nM 17β-Estradiol did not affect peak contraction in either sex but decreased the Ca
2+
transient in ♂ myocytes while increasing it in ♀ myocytes. DADLE and U50-488 had no effect on ♀ myocyte contractility but decreased ♂ myocyte contractility by a minimum of 13% and a maximum of 26% with/without 100 nM 17β-Estradiol. ♀ myocytes were immune to the effects of hypoxia, even exhibiting a slight increase in their peak calcium compared to control conditions, arguably because of the addition of isoproterenol. ♂ myocytes on the other hand, had an overall decrease in excitation-contraction coupling during hypoxia which was made worse by OR stimulation.

Conclusion:
♀ myocytes are immune to the detrimental effects of δ and κ OR stimulation.
Abstract 195: Epigenetic Mechanisms Underlying Anthracycline-Induced Cardiotoxicity
Authors: Chen Ching Kit , Lim Yee Phong , Tan Wilson Lek Wen , Anene-Nzelu George C , Ng Shi Ling , Li Yiqing , Danh Tuan L , Foo Roger Sik Yin
Published: October 2019

Anthracycline-induced cardiotoxicity (AC) is a major cause of morbidity in childhood cancer survivors. Doxorubicin cardiotoxicity however, is consistently observed to develop chronically over time. We sought to develop a mouse model of pediatric AC to test our hypothesis for an epigenetic mechanism, which may be operating to explain delayed-onset AC. Three week-old male and female C57Bl/6J mice were administered 5 weekly injections with doxorubicin (1mg/kg) (n=24) or saline (n=8). The dosage used was intended to closely mimic the clinical scenario. Half of the doxorubicin-treated (DOX) mice (n=12) and all controls (n=8) were sacrificed 1 week after the last injection; the rest of DOX mice (n=12) were given 4 weeks of drug-free holiday before sacrifice. Echocardiographic measurements were obtained before the first injection, 1 week after the final injection, and before sacrifice. Gene expression profiles were assessed by RNA-sequencing, reduced representation bisulphite sequencing (RRBS), and by ATAC-seq. These analyses were performed on purified pools of isolated cardiac myocytes, procured using a published Langendorff-free method. There was no death in the cohort. LV chamber dimension and wall thickness as well as fractional shortening did not differ between DOX and controls, implying the lack of obvious functional insufficiency from this dose. Similarly, DOX mice did not differ in body weight, heart weights (HW), and HW-to-tibia length ratios compared with controls. Cardiomyocyte dimensions and myocardial interstitial fibrosis were the same between groups. Despite the lack of echo and histological changes in DOX mice, RNA-seq analysis revealed profound and significant differentially expressed genes between the DOX mice and controls. Importantly, a significant proportion of downregulated and upregulated genes persisted, despite the period of drug-free holiday. Moreover, despite being taken off DOX, cardiomyocytes appeared to display new gene expression changes taking place even 4 weeks after stopping DOX. Detailed results of RNA-sequencing, RRBS and ATAC-Seq will be presented during the meeting. These results suggest that cardiomyocytes display DOX-related epigenetic alterations, which may implicate the basis for delayed-onset AC.
Abstract 224: Sub-cytotoxic Levels of Heavy Metals Induce Pro-inflammatory Signaling in the Aortic Endothelium without Impairing Flow-Mediated Dilation in Rats
Authors: Nabavizadeh Pooneh , Ibrahim Sharina , Fries Adam , Liu Jiangtao , Derakhshandeh Ronak , Springer Matthew L
Published: October 2019

Introduction:
Acute exposure to tobacco or marijuana secondhand smoke (SHS) causes endothelial dysfunction. The identity of specific SHS constituents that cause vascular toxicity is unclear. Heavy metals are present in SHS and at elevated levels in the blood of smokers, and may mediate acute endothelial dysfunction through reactive oxygen species formation and decreasing NO bioavailability. We assessed the effects of exposure to cadmium, lead, mercury, and arsenic at levels present in the blood of human smokers on flow-mediated dilation (FMD) as a measure of endothelial function in rats. We also evaluated the effects of heavy metal exposure on intimal structure, protein localization pattern, and inflammatory gene expression in the aortic endothelium.

Hypothesis:
Sub-cytotoxic levels of heavy metals impair FMD, alter intimal structure, and induce pro-inflammatory signaling in endothelium.

Methods:
We injected rats (n=8/group) with heavy metal cocktail or vehicle intravenously and quantitated pre- and post-exposure FMDs measures defined as percent vasodilation of femoral artery after transient ischemia. We performed en face aorta immunostaining and assessed endothelial cell axis alignment, cell length ratio, and localization pattern of PECAM-1, VE-cadherin, and vimentin. We also quantified gene expression of key endothelial proteins in aorta homogenates.

Results:
FMD was not impaired in the heavy metal group (8.8±3.6(SD)% vs. 12.9±8.0%, p=.31 or controls (7.5±2.7% vs. 8.8±5.8%, p=.63). No significant difference in cell length ratio and endothelial x and y axes of alignment were detected between groups (p>.8) and localization of PECAM-1, VE-cadherin, and vimentin in the aorta endothelium remained unaltered following heavy metal injection. However, expression of PECAM-1 and VE-cadherin was significantly lower in the heavy metal-treated rats, while VCAM-1 gene expression was significantly higher (p<.05).

Conclusion:
Acute exposure to sub-cytotoxic levels of heavy metals can induce pro-inflammatory signaling in endothelium, which could potentially lead to vascular injury. However, FMD and endothelial structure remain unchanged by heavy metal exposure.
Abstract 250: Smad3-Mediated Induction of Smad7 in Activated Myofibroblasts Protects the Remodeling Myocardium
Authors: Humeres Claudio D , Shinde Arti , Frangogiannis Nikolaos G
Published: October 2019

TGFβs are induced in the infarcted and pressure-overloaded myocardium and play an essential role in repair and remodeling by activating Smad-dependent and Smad-independent cascades. Fibroblasts are major targets of TGFβs. Activation of Smad3 signaling in cardiac fibroblasts is crucial in post-infarction repair, mediating formation of aligned myofibroblast arrays. TGFβ responses are tightly regulated through induction of endogenous suppressive signals, such as the inhibitory Smads, that prevent excessive TGFβ signaling. We hypothesized that the inhibitory Smad7 may be induced in activated myofibroblasts infiltrating infarcted and pressure-overloaded hearts, protecting the myocardium from adverse remodeling and fibrosis by restraining TGFβ actions.

Smad7 is markedly induced in infarcted and pressure-overloaded mouse hearts, and is localized in cardiomyocytes, myofibroblasts and activated macrophages. Induction of Smad7 in myofibroblasts is mediated through TGFβ/Smad3 signaling in vitro and in vivo. Mice lacking Smad7 in activated myofibroblasts (MFS7KO) and Smad7 fl/fl controls underwent non-reperfused myocardial infarction and transverse aortic constriction protocols. Following infarction, MFS7KO mice had higher mortality, worse systolic dysfunction and accentuated diastolic dysfunction when compared to Smad7 fl/fl. Following pressure overload, MFS7KO mice had accentuated interstitial and perivascular fibrosis and increased cardiomyocyte hypertrophy. In vitro, Smad7 overexpression attenuated myofibroblast conversion and reduced expression of profibrotic genes. Conversely, Smad7 knockdown promoted a matrix-synthetic fibroblast phenotype. Smad7 overexpression abrogated TGF-β-stimulated Smad2/3 and Erk activation without affecting TβRI and TβRII phosphorylation. Akt and p38 activation were unaffected by Smad7 overexpression. In conclusion, in remodeling hearts, Smad7 induction in activated myofibroblasts restrains fibrosis, negatively regulating Smad2/3 and Erk signaling through actions downstream of the TβRs. The protective effects of fibroblast Smad7 in cardiac remodeling may have important therapeutic implications for heart failure patients.
Abstract 251: Mitochondrial Calcium Uniporter Regulates Proliferative Activity of Cardiac Fibroblasts Under Angiotensin II Stimulation
Authors: Jhun Bong S , Suzuki Yuta , Cypress Michael W , Zhang Peng , Mende Ulrike , O-Uchi Jin
Published: October 2019

Introduction:
Cardiac fibrosis persists in patients with heart failure (HF) even during treatment with conventional HF therapies designed to control volume/pressure overload and restore cardiomyocyte function. This suggests a critical need to develop novel and effective anti-fibrotic therapies specifically targeting cardiac fibroblasts (CFs). CFs contribute to pathophysiological remodeling such as cardiac fibrosis after cardiac injury and/or during repair in response to various profibrotic stimuli
in vivo
such as angiotensin II (Ang II). Oxidative stress promotes pathological CF proliferation, but the detailed molecular mechanisms linking mitochondria and CF proliferation remain unclear.

Hypothesis:
Ang II stimulation promotes mitochondrial ROS (mROS) generation via mitochondrial Ca
2+
(mCa
2+
) uptake, which subsequently activates mROS-dependent proliferative signaling in CFs.

Methods:
In CFs isolated from rat hearts, mCa
2+
uptake, mROS levels and mitochondrial morphology were measured using confocal microscopy with a mitochondria-targeted Ca
2+
biosensor mtRCamp1h, a mitochondrial superoxide-sensitive dye MitoSOX, and mitochondria-targeted GFP, respectively..

Results:
Ang II (≥1 μM) stimulation induces Ca
2+
release from the endoplasmic reticulum (ER) followed by increased mCa
2+
, mROS, and mitochondrial fragmentation. In addition, Ang II activates ERK1/2- and p38-mediated proliferative pathways, which was inhibited by pretreatment with losartan, an Ang II receptor antagonist. Overexpression of a dominant-negative mCa
2+
uniporter (MCU) with pore domain mutations abolished Ang II-mediated mCa
2+
uptake and mROS generation, and prevented the activation of proliferative pathways. Pretreatment with a mitochondria-targeted antioxidant, mitoTEMPO, also significantly inhibited Ang II-mediated activation of the proliferative pathways without affecting ER-Ca
2+
release or mCa
2+
-uptake.

Conclusion:
The mROS generation via mCa
2+
accumulation and mitochondrial fragmentation resulting from Ang II stimulation activates mROS-dependent proliferative signaling pathways in CFs. The MCU-dependent mROS generation may serve as an important molecular switch for CF proliferation during cardiac stress.
Abstract 255: Novel Role for Free Fatty Acid Receptor 4 in Response to Pathologic Pressure Overload-Induced Heart Failure in Mice
Authors: Murphy Katherine A , Joshi Sonal S , Healy Chastity L , Ernste Katherine M , Harsch Brian A , Wagner Brandon M , Huang Wei , Jensen Brian C , Shearer Gregory C , O'Connell Timothy D
Published: October 2019

Free fatty acid receptor 4 (Ffar4, GPR120) is a GPCR for long chain fatty acids, including omega-3 polyunsaturated fatty acids (ω3-PUFAs). Previously, we demonstrated that eicosapentaenoic acid (EPA), an ω3-PUFA, prevents interstitial fibrosis and contractile dysfunction in pressure overload heart failure (transverse aortic constriction, TAC), but EPA was not incorporated into cardiac myocytes or fibroblasts, the traditional mechanism of action. Therefore, we hypothesized that Ffar4 is necessary for the anti-fibrotic and cardioprotective effects of EPA. In fact, we previously found that in primary cardiac fibroblasts, Ffar4 was sufficient and necessary to prevent TGFβ1-induced fibrosis. Here, we were surprised to find that Ffar4 also has important ω3-independent effects in cardiac myocytes
in vivo
. In mice with systemic deletion of Ffar4 (Ffar4KO mice) on a standard, corn oil diet (no EPA supplementation), TAC induced significantly more cardiac hypertrophy and more systolic (ejection fraction) and diastolic dysfunction (E/A ratio), but without excess fibrosis after 4 weeks. Mechanistically, transcriptome analysis in cardiac myocytes 3 days post-TAC indicated a deficiency of transcription in Ffar4KO myocytes. Sorting based on gene ontology indicated that genes functionally categorized as cell death and inflammation, including
ilrn1
,
hmox1
,
Il33
, and
ptgs2
(cox2) were not induced in the Ffar4KO myocytes. Ffar4 also activates cytoplasmic phospholipase A2 (cPLA2) inducing the production of oxylipins, and systemic deletion of cPLA2 induces exaggerated hypertrophy during development and following TAC. TAC increased cPLA2 expression in WT, but not Ffar4KO cardiac myocytes and oxylipin production was reduced in Ffar4KO mice. Finally, we confirmed that Ffar4 is expressed in human heart and down-regulated in heart failure. In summary, our data identify a novel, ω3-independent cardioprotective role for Ffar4 in cardiac myocytes.
Abstract 264: EPRS is a Critical Translational Control Factor in Cardiac Fibrosis
Authors: Wu Jiangbin , Venkata Subbaiah Kadiam C , Jiang Feng , Hedaya Omar , Yao Peng
Published: October 2019

Increased protein synthesis of pro-fibrotic genes is a common feature of cardiac fibrosis, a major manifestation of heart failure (HF). Despite this observation, critical factors and molecular mechanisms for translational control of cardiac fibrosis remain underexplored. Here, we identify a critical house-keeping translational regulator, glutamyl-prolyl-tRNA synthetase (EPRS), which preferentially regulates the translation of proline-rich (PRR) pro-fibrotic genes. In mammals, EPRS catalyzes the attachment of two amino acids, glutamic acid and proline, to their cognate tRNAs for protein synthesis. By overlapping of aminoacyl-tRNA synthetases (ARSs) induced in TGF-β treated human cardiac fibroblasts, human ARSs with genetic mutations in the congenital heart disease, mouse ARSs associated with isoproterenol (ISO)-induced cardiomyopathy by GWAS, and ISO-induced ARSs in mouse failing hearts, we identified EPRS as the only ARS involved in various cardiac pathogenesis. EPRS is induced in failing human heart compared to non-failing donor heart and functions as an integrated node downstream of multiple hypertrophic and fibrotic stimuli in murine hearts, including ISO infusion and transverse aortic constriction (TAC) surgery. Low-dose halofuginone (Halo), a prolyl-tRNA synthetase (PRS)-specific inhibitor, as well as genetic knockout of one allele of EPRS in mouse genome, reduces cardiac hypertrophy and fibrosis in ISO- and TAC-induced HF mouse models. Using RNA-Seq and polysome profiling-Seq in Halo-treated fibroblasts, we identified several novel PRR genes, including Ltbp2, Furin and Sulf1, in addition to collagens, which are translationally regulated by EPRS. Inhibition of Furin by various inhibitors attenuates cardiac fibroblast activation and collagen deposition
in vitro
. Finally, we found that inactivation of EPRS reduced translational efficiency and enhanced mRNA decay of PRR genes. Taken together, our results indicate that EPRS controls the translational activation of PRR genes in cardiac fibroblasts, and these data provide novel insights into the translational control mechanisms of cardiac fibrosis, which may promote development of novel therapeutics by inhibiting pro-fibrotic translation factors.
Abstract 265: Thyrotoxic Pericarditis in a Mouse Model of Graves' Disease
Authors: Zhao Fengyi , Wu Liping , Shi Bingyin
Published: October 2019

Objectives:
To investigate the cardiac pathological changes of hyperthyroid heart disease in mice.

Methods:
A transient model for human Graves’ disease was successfully established in mice using three immunizations with recombinant adenovirus expressing the extracellular A subunit of the human thyrotropin receptor (Ad-TSHR). We induced the hyperthyroid heart mouse model by prolonging the regular injections of Graves’ disease mouse model to nine months. Heart tissues were weighed and stained with hematoxylin-eosin (HE) and MASSON Trichrome staining to check and define the pathological changes.

Results:
We observed that mice in the Graves’ disease hyperthyroid heart group (GH) showed enhanced cardiac hypertrophy and fibrosis. The HW/BW (ratio of heart weight to body weight) of GH group (5.528±0.4014, n=6) was significantly higher than control group (4.343±0.03913, n=6) (P<0.05). In GH group, 2 of 6 hearts showed typical cor villosum (Image 1). The MASSON Trichrome staining exhibited aggravated cardiac fibrosis on the surface of the heart in GH group.

Conclusions:
Pericarditis as a complication of thyrotoxicosis has been reported several times in clinical work. This is the first reported case of pericarditis in the mouse model of Graves’ disease, suggesting a possible link between pericarditis and thyrotoxicosis.
Abstract 277: Neonatal Cardiomyocyte-Released Signaling Protein Reduces Adipocyte Differentiation
Authors: Gresham Kenneth S , Koch Walter J
Published: October 2019

The heart continuously cycles through periods of muscle contraction and relaxation to pump blood throughout the body, which requires a constant supply of metabolic substrates to the heart to power these contractile cycles. To ensure adequate energy availability, the heart must communicate with other organ systems to maintain global metabolic energy homeostasis. In addition to the role of the sympathetic nervous system in global metabolic regulation, mounting evidence suggests cardiomyocytes can release signaling factors that alter global energy balance through communication with other organ systems, including the liver and adipose tissue. To identify potential cardiomyocyte-released circulating factors, we collected media conditioned by neonatal rat ventricular myocytes (NRVM-CM) and screened for released-factor activity using an
in vitro
adipocyte differentiation assay. Treatment of differentiating 3T3-L1 adipocytes reduced lipid accumulation and decreased expression of adipocyte proteins, demonstrating NRVM-released factor activity. Released factor activity was also under the control of regulators of GPCR signaling as overexpression of G-protein coupled receptor kinase 2 (GRK2) further enhanced the NRVM-released factor activity. We further determined that NRVM released-factor activity was abolished by trypsin treatment and could be segregated into >30kDa CM fractions using centrifugal filter units. To identify proteins responsible for the NRVM-released factor activity, NRVM-CM retained in the 30kDA centrifugal filter units (containing all proteins >30kDa) was analyzed by mass spectrometry. This revealed a number of signaling proteins with known adipocyte regulatory roles that could potentially regulate adipocyte physiology. We confirmed these results by examining NRVM cell lysate and concentrated NRVM-CM by Western blot to test for the presence of decorin, one of the proteins identified by mass spectrometry known to have adipocyte regulatory roles. These results provide a framework to identify potential
in vivo
cardiomyocyte-released factors that regulate global energy balance.
Abstract 284: Drp1 Protects the Heart Against High Fat Diet-Induced Diabetic Cardiomyopathy
Authors: Tong Mingming , Zhai Peiyong , Mizushima Wataru , Sadoshima Junichi
Published: October 2019

Type II diabetes induces cardiomyopathy characterized by hypertrophy, diastolic dysfunction and mitochondrial dysfunction. We have shown recently that clearance of damaged mitochondria by mitophagy mediated through an Atg7-dependent mechanism serves as an essential mechanism to maintain the quality of mitochondria during the initial development of diabetic cardiomyopathy. Our previous study showed that disruption of Drp1 inhibits mitophagy and causes mitochondrial dysfunction at baseline and in response to pressure overload. We therefore investigated whether Drp1 is essential for mitophagy during high fat diet (HFD)-induced diabetic cardiomyopathy. Mice were fed either a normal diet (ND) or a HFD (60 kcal % fat). Mitophagy, evaluated with Mito-Keima, was increased after 3 weeks of HFD (p<0.05). However, this increase in mitophagy induced by HFD consumption was abolished in tamoxifen-inducible cardiac-specific Drp1 knockout (Drp1 cKO) mouse hearts (p<0.05), in which both diastolic dysfunction (p<0.05) and systolic dysfunction (p<0.05) were exacerbated. Furthermore, the increase in LC3-dependent general autophagy in response to HFD consumption was also abolished in Drp1 cKO mice (p<0.05). Electron microscopic analyses showed elongated mitochondria and accumulation of lipid droplets in Drp1 cKO mice (p<0.05) in the presence of HFD consumption. Fibrosis and cell death were increased in Drp1 cKO mice during HFD consumption (p<0.05). Thus, Drp1 is essential for mitophagy during HFD consumption and serves as an essential mitochondrial quality control mechanism, thereby protecting the heart against diabetic cardiomyopathy.
Abstract 289: Neddylation is Essential for Cardiac Metabolic Maturation in the Developing Heart
Authors: Zou Jianqiu , Li Jie , Littlejohn Rodney , Yao Yali , Dong Kunzhe , Zhou Jiliang , Chen Weiqin , Su Huabo
Published: October 2019

Changes in metabolic milieu during development trigger a metabolic shift from fetal-type glycolysis to adult-type fatty acid oxidation in the developing heart. Disruption of this metabolic shift has been linked to congenital heart disease, a major cause of birth-related mortality and morbidity worldwide. Neddylation is a post-translational modification that covalently attaches a small ubiquitin-like protein, NEDD8, to target proteins. Previously we reported that neddylation is essential for cardiomyocyte proliferation through sustaining the YAP signaling. However, a link between neddylation and cardiac metabolic maturation has not yet been established. Here, we abrogated neddylation by cardiac-specific deletion of NAE1, an essential subunit of the sole NEDD8 E1 enzyme, via αMHC
Cre
in mice, which led to significant reduction of NAE1 proteins and neddylated proteins in the heart. Temporal histological and functional analyses revealed that mice lacking neddylation displayed cardiac hypoplasia and ventricular non-compaction at E16.5, which became more pronounced by P1, eventually leading to heart failure and perinatal lethality by P7. Transcriptome analysis identified that the defects in cardiac chamber maturation are associated with upregulation of glycolytic genes and downregulation of oxidative metabolic genes. Transmissive electron microcopy revealed accumulation of lipid droplets and degenerative/immature mitochondria in hearts deficient of neddylation. In vitro, pharmacological inhibition of neddylation impairs mitochondrial membrane potential and respiration, and suppresses fatty acid utilization in oleic acid-primed cultured cardiomyocytes. Mechanistically, HIF1α, a potent regulator of cardiac metabolic reprogramming, is a novel NEDD8 target. Inhibition of neddylation causes accumulation of HIF1α proteins and consequently disrupts the expression of HIF1α downstream targets in vitro and in vivo. Silencing of HIF1α attenuates inhibition of neddylation-induced glycolytic rates in cardiomyocytes. Taken together, our findings highlight the importance of neddylation in the developing heart and identify neddylation as a novel regulator of HIF1α signaling to promote developmental metabolic switch.
Abstract 300: Ischemia and Reperfusion Injury Following Cardioplegic Arrest is Attenuated by Age and Testosterone Deficiency in Male but Not Female Mice
Authors: Ghimire Anjali , Bisset Elise S , Howlett Susan E
Published: October 2019

Cardiovascular disease increases with age in both sexes and treatment can require cardiac surgery, where the heart is pre-treated with a protective cardioplegic solution before exposure to ischemia and reperfusion (I/R). While endogenous estrogen is thought to be beneficial in I/R, the role of testosterone in cardioprotection is not well understood and whether age modifies responses to I/R is unclear. We investigated sex- and age-specific differences in I/R injury in hearts pre-treated with a clinically relevant cardioplegic solution. Hearts were isolated from adult (6-9 mos) and old (22-28 mos) C57BL/6 mice of both sexes (n=27). They were Langendorff-perfused with Krebs-Henseleit buffer (15 min; 37°C), followed by St. Thomas’2 cardioplegia (6 min; 6-7°C). This was followed by 90 min of global ischemia (room temperature) and 30 min of reperfusion (37°C). Hearts were then perfused with triphenyltetrazolium chloride to quantify infarct area. The role of testosterone was investigated in gonadectomized (GDX; 6-9 mos) male mice and serum testosterone was measured with an ELISA. Functional recovery in reperfusion was significantly better in old males than in adult males. Left ventricular developed pressure (LVDP) recovered to 67.3 ± 7.4% in old compared to 31.6 ± 12.3% in adult male hearts (p<0.05). Similar results were seen for rates of pressure development (+dP/dt; p<0.05) and decay (-dP/dt; p<0.05). Also, infarct areas were markedly smaller in old male hearts (15.5 ± 1.8%) than in younger hearts (48.1 ± 7.7%; p<0.05). By contrast, hearts from adult and old females exhibited a similar degree of post-ischemic functional recovery and showed no age-dependent difference in infarct size. Serum testosterone levels declined with age in males but were low regardless of age in females, which suggested a potential benefit of low testosterone. In support of this, hearts from GDX males exhibited much better recovery of LVDP in reperfusion when compared to hearts from intact males (values were 64.8 ± 7.7% vs. 29.9 ± 12.3%; p<0.05). GDX hearts also had smaller infarcts than hearts from intact males (p<0.05). These results suggest that low testosterone levels may be protective against I/R injury following cardioplegic arrest in the setting of aging, especially in old males.
Abstract 308: Role of Snf1-related Kinase as a Regulator of Chromatin Modifications and Dna-damage Response in Heart Injury
Authors: Stanczyk Paulina J , Zilber Zachary , de Jesus Adam , Chang Hsiang-Chun , Ardehali Hossein
Published: October 2019

Introduction:
Altered DNA damage response (DDR) can result in phenotypic changes that share features with age-associated pathological conditions, including metabolic and cardiovascular abnormalities. Snf1-related kinase (SNRK) is a serine/threonine kinase that belongs to the AMPK family. Our recent data demonstrates that SNRK expression is upregulated in heart failure (HF) and low energy states and that SNRK improves cardiac mitochondrial efficiency and cell survival. Here, we assessed the hypothesis that in response to stress, SNRK translocates to the nucleus where it modulates nuclear processes to enhance DNA repair and ultimately reduces cardiomyocyte death.

Results:
We first showed that the nuclear distribution of endogenous SNRK in cardiomyoblast is enhanced under low nutrient and oxidative stress. Next, we performed global phosphoproteomics analysis on cells with overexpression of wild type SNRK and mutant kinase-dead SNRK (D158A). We found significant increase of several phosphopeptides involved in nuclear processes, in particular in the regulation of chromatin remodeling and DDR. SNRK downregulation resulted in an increase in nucleus volume and heterochromatin content, with increased nuclear dysmorphia when subjected to oxidative stress. Despite having lower expression of DDR signal effectors p53 and Mdm2, these cells accumulated higher levels of pH2AX (DDR marker) and displayed higher cellular death after oxidative stress, consistent with increased DNA damage. Etoposide treatment in SNRK knockdown cells failed to induce p53 levels despite pH2AX accumulation. Additionally, pharmacological stabilization of p53 protein through nutlin-3A treatment did not increase p53 in SNRK knockdown cells. These findings suggest that SNRK regulates p53 expression, thereby acting as a new modulator of DDR.

Conclusions:
Our results demonstrate increased nuclear localization of SNRK under stress conditions, and that SNRK regulates phosphorylation of nuclear proteins involved in chromatin remodelling and DDR. Thus, SNRK may induce its pro-survival cardiac functions through regulation of chromatin architecture and enhancing DDR. Those novel findings can contribute to development of new therapies against HF.
Abstract 310: Time-Dependent Changes in Myocardial Edema Overestimate the Ischemic Area-at-Risk Up to One Week After Reperfused Myocardial Infarction in Swine
Authors: Weil Brian R , Techiryan George , Canty John M
Published: October 2019

Objective:
Assessment of tissue salvage after myocardial infarction (MI) requires accurate quantification of the ischemic area at risk (AAR). Although the AAR is typically measured retrospectively within one week after reperfusion, dynamic changes in tissue composition may affect the precision of this approach. Given recent cardiac MRI data demonstrating a transient reduction in myocardial edema 24 hours after reperfused MI, we tested the hypothesis that this is an optimal timepoint for accurate retrospective AAR assessment.

Methods:
Swine (n=27) were subjected to a 1 hour LAD occlusion, during which the AAR was assessed by contrast enhanced CT-derived quantification of the myocardial perfusion defect. Phthalocyanine blue was administered at the end of occlusion (n=7) or during re-occlusion of the LAD 3 hours (n=5), 24 hours (n=5) or 7 days (n=10) after reperfusion for pathologic determination of the AAR. Serial echocardiography was performed in the 24 hour reperfusion group to assess end diastolic wall thickness (EDWT) as a surrogate index of post-MI edema.

Results:
When assessed before reperfusion, the AAR was similar by CT and postmortem pathology (
Table
). However, AAR measurements 3 hours, 24 hours, and 7 days after reperfusion overestimated CT AAR mass by 38±7 %, 31±10 %, and 37±5%, respectively. Echocardiography showed a marked increase in EDWT 1 and 3 hours after reperfusion, followed by a decrease at 24 hours that remained significantly higher than pre-ischemia values.

Conclusion:
Despite partial resolution of edema 24 hours after reperfusion, AAR measurement at this time does not provide an accurate assessment of the AAR obtained during the initial coronary occlusion.
Abstract 335: Titin Truncations in Human Heart Tissue
Authors: McAfee Quentin , Brandimarto Jeff , Rhoades Joshua , Bede Ken , Margulies Kenneth , Arany Zoltan
Published: October 2019

Truncating variants in the gene for titin (TTNtvs) strongly associate with dilated cardiomyopathy (DCM) and peripartum cardiomyopathy (PPCM). How TTNtvs lead to cardiomyopathy is unclear. Both haploinsufficient and dominant negative mechanisms have been proposed. To begin to address this question, we have obtained left ventricular myocardium from explanted hearts from patients with DCM or PPCM undergoing cardiac transplantation, as well as myocardium from non-failing donor hearts. Targeted exome sequencing identified 8 hearts bearing TTNtvs in isoforms expressed in adult heart. Modified gel electrophoresis of extracts from these samples revealed no apparent band at the expected (truncated) molecular weight predicted for each TTNtv, indicating that truncated titin protein is likely absent. In addition, the total amount of titin protein appeared preserved in TTNtv extracts, arguing against haploinsufficiency in these hearts. RNAseq of TTNtv-positive hearts revealed balanced representation of all identified heterozygous variants, i.e. preserved allelic balance, indicating that the alleles encoding TTNtvs are appropriately transcribed, and do not undergo nonsense mediated decay. Together these data support neither haploinsufficiency nor dominant negative mechanisms, and leave open the question of how TTNtvs contribute to the development of DCM and PPCM.
Abstract 336: The Effect Of Phosphodiesterase 9a Inhibition In 2 Mouse Models Of Diastolic Dysfunction
Authors: Methawasin Mei , Strom Josh , Hourani Zaynab , Smith John E , Granzier Henk L
Published: October 2019

Low myocardial cGMP-PKG activity is associated with increased cardiomyocyte stiffness in HFpEF. cGMP is mainly hydrolyzed by PDE (phosphodiesterases)5a and PDE9a. Importantly, PDE9a protein expression was reported to be upregulated in human HFpEF myocardium and chronic administration of PDE9a inhibitor reversed pre-established cardiac hypertrophy and systolic dysfunction in mice subjected to TAC (Transverse Aortic Constriction).

We evaluated whether PDE9a inhibition had a beneficial effect on diastolic dysfunction (DD). We investigated two mouse models of DD: 1) TAC-DOCA (TAC with deoxycorticosterone acetate (DOCA) supplementation) and 2) LepR
db/db
, a metabolically induced DD models. Mice were subjected to subcutaneous implantation with osmotic minipumps containing either PD9a inhibitor (PF-4449613) or vehicle for 4 wks. PDE9a transcript was not upregulated in myocardium of either TAC-DOCA or LepR
db/db
mice. PV analysis revealed that in TAC-DOCA mice, inhibition of PDE9a resulted in reduced LV diastolic stiffness, but impairment of systolic function observed as ventricular-vascular decoupling. In LepR
db/db
mice, cardiomyocyte stiffness was slightly but significantly reduced in PDE9a inhibitor treated animals. No differences in myocardial fibrosis or cardiac morphometry were observed.

The effect of acute PDE9a inhibition was investigated in intact cardiomyocytes isolated from LVs of TAC-DOCA mice. Cellular work loop force measurement was conducted before and after administration of PDE9a inhibitor combined with ANP (Atrial Natriuretic Peptide) for 15 mins. Cardiomyocytes from TAC-DOCA had high diastolic stiffness compared to sham hearts, however, no stiffness reduction was observed after acute inhibitor treatment.

Conclusion:
Chronic inhibition of PDE9a ameliorates LV diastolic stiffness, but suppresses systolic function in TAC-DOCA induced DD mice. The LV stiffness reduction is in part due to cardiomyocyte stiffness reduction. The advantage of PDE9a inhibition on diastolic function might be more evident, with less systolic suppression, in hearts with PDE9a upregulation.
Abstract 338: Motivating Myosin: 2 deoxy-ATP Induced Structural Alterations That Increase Myosin Activity
Authors: Regnier Michael
Published: October 2019

When 2-deoxy-ATP (dATP) replaces ATP as the contractile substrate, cardiac myosin binding to actin and the rate of cross-bridge cycling is increased. To understand the structural basis of this we employed low-angle x-ray diffraction and computational modeling. Molecular Dynamics (MD) simulations of pre-powerstroke myosin suggest dADP.Pi (vs. ADP.Pi) induces changes in contact pairs for nucleotide binding that translate to structural changes on the actin binding surface, exposing positive charge in regions that make electrostatic interactions with actin. Brownian Dynamics simulations suggest M.dADP.Pi binds more rapidly to actin at distances (angstrom) associated with weak binding. X-ray diffraction analysis of resting demembranated cardiac muscle indicated a large increase in the I
1,1
/I
1,0
intensity ratio for M.dADP.Pi, suggesting myosin moves towards thin filaments. This difference was eliminated at low ionic strength (100 vs. 170mM), where more protein surface charge is exposed, thus decreasing the electrostatic interaction advantage of M.dADP.Pi. The S
M3
meridional reflection indicated spacing between myosin crowns was increased for M.dADP.Pi at rest, making it similar was to the activated position of M.ADP.Pi during contraction, with no change in axial ordering (
I
M3
).In intact soleus muscle of transgenic mice with 1% dATP (99% ATP), time-resolved x-ray structure indicates myosin dissociation from actin following tetanic contraction is slightly slower (vs WT) bu the first-order myosin layer line (
MLL1
) intensity recovers faster, suggesting dATP increases the rate myosin heads return to an ordered resting state. At rest the radii to the center of mass of myosin heads (R
m
) and I
1,1
/I
1,0
were larger, indicating myosin heads were closer to actin. MD simulations of post-powerstroke myosin showed greater dADP mobility in the binding pocket via altered interactions with key amino acids, resulting in altered conformation of residues on the actin binding surface in regions interacting with actin. Combined our results suggest that with dATP myosin moves closer to actin and S1 heads are more activated in resting muscle, staying primed for reactivation following relaxation, all via increased myosin-actin electrostatic interactions.