bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 507: Activation of Transient Receptor Potential Canonical Channel Currents in Iron-Overloaded Cardiac Myocytes
Authors: Siri-Angkul Natthaphat , Gordan Richard , Wongjaikam Suwakon , Fefelova Nadezhda , Gwathmey Judith , Chattipakorn Siriporn , Chattipakorn Nipon , Xie Lai-Hua
Published: October 2019

Background:
Iron (Fe) overload cardiomyopathy is the leading cause of death in hemochromatotic patients, yet the mechanistic insight is still incomplete and controversial. We investigated alterations of action potentials (APs), ionic currents, and intracellular Ca
2+
(Ca
2+
i
) in Fe-loaded mouse cardiomyocytes, as well as functional impacts of Fe overload on single-cell contraction and whole-heart arrhythmias.

Methods:
Cardiomyocytes were isolated from left ventricles of mouse hearts and were superfused with Fe
3+
/8-hydroxyquinoline complex (5-100 μM). APs, L-type Ca
2+
currents (I
Ca,L
), total outward K
+
currents (I
K
), and transient receptor potential canonical (TRPC) channel currents were recorded by the patch-clamp technique. Ca
2+
i
was evaluated by using Fluo-4. Cell contraction was measured by a video-based edge detection system. Arrhythmias were evaluated in Langendorff-perfused hearts under S
1
-S
2
stimulation protocol.

Results:
Persistent Fe (15 μM) treatment prolonged AP duration at 90% repolarization (APD
90
: 46.8 ± 2.8 vs. 203.6 ± 63.4 ms, p<0.05), induced early and delayed afterdepolarizations (EADs: 0 % vs. 45.0 ± 15.0 %; DADs: 4.3 ± 1.4 vs. 27.0 ± 7.0 %, p<0.05, respectively) in mouse cardiomyocytes. Consistently, arrhythmia incidence was increased in Fe
3+
/8-HQ-perfused hearts. Fe treatment decreased peak I
Ca,L
(16.5 ± 1.7 vs.11.4 ± 1.3 pA/pF, p<0.01) and I
K
(59.2 ± 3.3 vs. 50.4 ± 3.0 pA/pF, p<0.01), altered Ca
2+
i
transient patterns and decreased contractility (4.8 ± 0.5 vs. 3.5 ± 0.4%, p<0.01). During the late phase of Fe treatment, fast Ca
2+
waves and sustained depolarization were induced to generate a secondary (shallow) resting membrane potential (RMP: from -68.8 ± 0.6 to -25.0 ± 3.7 mV) where the myocytes became unexcitable. Gadolinium, a TRPC channel blocker, abolished fast Ca
2+
waves and reversed RMP to the deep level (-62.9 ± 3.5 mV). The involvement of TRPC activation was determined for the first time by recording TRPC current and assessing the effect of functional TRPC channel antibodies.

Conclusions:
In mouse cardiomyocytes, Fe overload induced arrhythmogenic APD prolongation and EADs/DADs, aberrant Ca
2+
i
dynamics, and impaired contractility. The activation of TRPC channels accounts for an important underlying mechanism.
Abstract 514: Proteomic Characterization of Extracellular Matrix Regulation in Human Aortic Valve Development and Disease
Authors: Clift Cassandra L , Bethard Jennifer , Comte-Walters Susana , Ball Lauren E , Bichell David , Su Yan R , Mehta Anand , Drake Richard R , Angel Peggi M
Published: October 2019

We present the first report of the collagen protein interactome in human aortic valve (AV) development and disease. AV disease affects up to 13% of the world population. As no therapies are available, patients must “watch and wait” until surgical valve replacement is necessary to prevent heart failure. Extracellular matrix (ECM) forms the basis for correct valve function but alterations of the ECM scaffold during development and disease are not well defined. Here, we report ECM protein regulation from a cohort of 26 valvular tissues in four pathological categories: normal functioning AVs, pediatric end-stage congenital aortic valve stenosis (CAVS), adult end-stage fibrocalcific aortic valve stenosis (FAVS), and a pulmonic Ross operation valve. A novel proteomic method was used to target ECM proteins, primarily collagens, for sequence analysis from FFPE 5-μm thin tissue sections. Collagens represented 19/49 proteins identified (39%); A total of 46/49 ECM proteins (94%) were implicated in direct interactions in collagen synthesis, regulation or modification. Data suggested dynamic regulation of collagen types during age and disease, e.g., Col15a1 was found only in normal AV, whereas Col4a1 was reported only in patients younger than 5 years. Upstream regulators of the collagen interactome included COLQ1, IGFBP2, and SPARC. Detailed peptide sequence analysis reported 70 specific collagen peptide sequences containing up to multiple hydroxylated prolines (HYP), a post-translational modification critical to stabilizing the collagen triple helix. Quantitative data analysis on HYP peptides reported differential regulation across patient categories – i.e., Col3a1 peptides show 52% increased HYP in CAVS compared to age matched normal valve. Interestingly, the pulmonic Ross valve represented unique collagen HYP signatures distinct from all other aortic valve tissues. Tissue from pediatric end-stage CAVS patients showed a higher percentage of hydroxylated peptides compared to non-hydroxylated peptides (33%) as compared to normal (25%) or adult end-stage FAVS (16%). We anticipate that the completed study will be useful to targeted therapies aiming to inhibit fibrosis and ECM remodeling, and may better inform engineered options for valve replacement.
Abstract 520: Cavin-2/SDPR in Cardiac Fibroblasts Modulates TGF-β/Smad Signaling and Promotes Pressure Overload-induced Fibrosis
Authors: Higuchi Yusuke , Ogata Takehiro , Nakanishi Naohiko , Nishi Masahiro , Sakamoto Akira , Tsuji Yumika , Matoba Satoaki
Published: October 2019

Introduction:
Heart failure (HF) is a debilitating disease associated with high morbidity and mortality. The high mortality rate reflects the inadequacy of modern therapy and calls for new treatments. A major cause of HF is the adverse tissue remodeling with fibrosis. Excessive extracellular matrix (ECM) turnover is involved in the poor outcome. Resident fibroblasts are responsible for cardiac fibrosis on pressure-overload heart failure. Caveolins and Cavins are known as caveolar-related proteins. Caveolin-1 inhibits TGF-β1-induced fibrosis in the previous reports. Although Cavin-2/Serum deprivation response protein (SDPR), which is one of the caveolar-relatedproteins, is also abundant in fibroblasts, the role of Cavin-2 in cardiac fibrosis and function remains unknown.

Methods and Results:
To clarify the role of Cavin-2 in the cardiac fibroblasts in the pressure-overloadedheart, we performed transverse aortic constriction (TAC) operation on SDPR
flox/flox
mice and fibroblast-specific Cavin-2 cKO mice (Postin
Cre
; SDPR
flox/flox
). Four weeks after TAC, left ventricular fractionalshortening (LVFS) was preservedwith a significant reduction of cardiac fibrosis in PostinCre; SDPRflox/flox mice. Fibrosis-associated mRNA expressions (
Col1a1
,
Ctgf
,
Col3
), α1 type I collagen deposition, and αSMA-positive cells were also reducedin the hearts of Cavin-2 cKO mice after TAC. Trans-differentiation of fibroblasts into activated myofibroblasts is a defining feature of fibrosis. Myofibroblasts express α-smooth muscle actin (αSMA) and secrete ECM proteins via Smad signaling.
In
mouse embryonic fibroblasts (MEFs),Cavin-2 deficiency reduced the levels of αSMA protein and fibrosis-associated mRNA expressions by TGF-β1 stimulation. Furthermore, TGF-β1-induced Smad2 phosphorylation was attenuatedin Cavin-2 KO MEFs compared to WT MEFs. On the other hand, adenovirus-mediated Cavin-2 overexpression significantly increased αSMA and the fibrosis-associated mRNA expressions.

Conclusions:
Our observations suggest that Cavin-2 contributes to the development of cardiac fibrosis through the differentiation from fibroblasts into myofibroblasts via TGF-β/Smad signaling. Cavin-2 may be a novel therapeutic target for cardiac fibrosis.
Abstract 531: Urolithin a Suppress Cardiac Fibrosis via Autophagy Pathway in the Diabetic Cardiomyopathy
Authors: Wu Xiaoqian , Zhu Xiaoyan , Zhou You
Published: October 2019

Objective:
The cardiovascular protective effect of Urolithins remains poorly understood, despite wide-spread human exposure via the dietary consumption of their metabolic precursors, the ellagitannins, which is found in the pomegranate fruit. We identified Urolithin A (UA) as a natural compound that induces mitophagy and protects against diabetic cardiomyopathy both in vivo and in vitro. Healthy male C57BL/6 mice were fed with a High-fat diet for 6 months combined with a small dose of STZ injection to establish the T2DM model. Compared with the control, the diabetic mice suffered from cardiac dysfunction characterized by decrease EF, FS and E/A ratio, which was reversed with UA treatment. Masson staining and collagen marker Immunoblots showed that cardiac fibrosis was also decreased with UA treatment in the diabetic mice both in vivo and in vitro. Interestingly, we found UA significantly activated mitophagy and increased the co-localization of Myto-tracker and Lysotracker. However, we found UA increased Drp1 expression but not PINK1 or Parkin phosphorylation indicating a non-canonical mitophagy pathway. We treated the cardiac fibroblast with Drp1 siRNA and found the protective effect of UA against cardiac fibrosis was abolished.

Conclusion:
UA protected against diabetic cardiomyopathy by activating mitophagy vias a non-canonical pathway. Keywords: Diabetic cardiomyopathy; Cardiac fibrosis; Urolithin A; Autophagy; Mitophagy
Abstract 547: Metabolomic and Transcriptomic Profiling of Rat Offspring Exposed to Gestational Diabetes Reveals Altered Cardiac Gene Expression and Metabolism
Authors: Kereliuk Stephanie , Agarwal Prasoon , Cole Laura , Cheung Kyle , Xiang Bo , Fonseca Mario , Hatch Grant , McGavock Jonathan , Dolinsky Vernon
Published: October 2019

Gestational diabetes mellitus (GDM) is the most common complication of pregnancy. Children exposed to GDM are at an increased risk of developing cardiometabolic diseases later in life, though the mechanisms responsible are unknown. We hypothesize that fetal exposure to GDM induces alterations in cardiomyocyte metabolism and concomitant left ventricular (LV) dysfunction with age. GDM was induced in female rats with a high fat (45% kcal) and sucrose diet prior to mating, throughout pregnancy and lactation. Lean control females received a low fat (10% kcal) diet. Fetal rat ventricular cardiomyocytes (FRVC) were isolated from e20.5 offspring for U-13C glucose metabolic flux analysis and mitochondrial respiration. Serum metabolites and cardiac transcriptome profiles were measured in 3-month old offspring. LV morphology and function was assessed through the life course of the offspring (e18 to 12-months of age) by transthoracic ultrasound. Offspring exposed to GDM exhibited increased LV posterior wall thickness across their life course (fetal to 12-months of age; p<0.05) and impaired LV filling beginning at 6-months of age (p<0.05). U-13C glucose metabolic flux through glycolysis and the citric acid cycle was reduced in FRVC from GDM offspring when treated with isoproterenol, and compared to Lean FRVC. Basal and maximal mitochondrial oxygen consumption was reduced for glucose (35% and 68%) and fatty acid (49% and 52%) substrates in FRVC isolated from GDM offspring (p<0.05). In 3-month old GDM offspring, serum metabolomics revealed elevated levels of beta-hydroxybutyrate (2.4-fold, p<0.05) and reduced levels of several citric acid cycle intermediates, which also corresponded to alterations in gene expression patterns identified by RNASeq transcript analysis. Large-scale profiling revealed GDM induced alterations in the cardiac gene expression profile leading to modified serum metabolite levels in the offspring. These alterations corresponded with mitochondrial dysfunction, impaired cardiomyocyte metabolic flux and contractility, in concert with LV hypertrophy and diastolic dysfunction in the rat offspring. Our findings identify several mechanisms that link early-life GDM exposure to the development of cardiovascular disease later in life.
Abstract 549: Cardiac Aging is Associated With Impaired Mitophagy and Formation of Megamitochondria
Authors: Liang Wenjing , Moyzis Alexandra G , Najor Rita A , Lampert Mark A , Gustafsson Åsa B
Published: October 2019

Aging is associated with increased risk of developing cardiovascular disease. Currently, the molecular mechanisms contributing to the cardiac aging process and the resulting pathogenesis are unclear. Here, we have characterized changes in mitochondria and mitochondrial autophagy (mitophagy) in aging mouse hearts. To evaluate structural and functional changes that occur with cardiac aging, we compared young (4 months) and old (24 month) mice. We found that aging led to cardiac hypertrophy as characterized by increased left ventricular (LV) mass index and heart weight/body weight ratio as well as elevated myosin heavy chain 7 mRNA levels. Although echocardiography analysis showed no detectable changes in systolic function, the aged mice showed evidence of diastolic dysfunction as there was a significant decrease in the E/A ratio. We also found increased levels of fibrosis and inflammation in the aged hearts. Evaluation of autophagy in aged hearts revealed impaired autophagic flux, as indicated by accumulation of the autophagy substrate p62. Treatment of mice with the mTOR inhibitor Rapamycin, led to the expected increase in LC3II-positive autophagosomes in young hearts but not in the aged hearts. Parkin is an E3 ubiquitin ligase involved in labeling mitochondria for mitophagy by ubiquitinating proteins on the outer mitochondrial membrane. We found a significant increase in Parkin at the mitochondria in aged hearts which was accompanied with a significant increase in mitochondrial protein ubiquitination and p62 levels. However, LC3II protein levels in the mitochondrial fraction did not increase. This suggests that although mitochondria have been labeled for mitophagy, they are not eliminated due to decreased formation of autophagosomes. Finally, ultrastructural analysis of hearts showed the presence of enlarged mitochondria in aged hearts. Although we observed no changes in mitochondrial fusion proteins Mfn1/2, we found decreased activation of the mitochondrial fission protein Drp1. Overall, these data suggest that which might contribute to the development of age-related cardiac pathologies.
Abstract 588: Beta-blockers Reverse Beta Adrenergic Receptors Desensitization Under Hypoxia
Authors: Sun Yu , Gupta Manveen , Stenson Kate , Prasad Sathyamangla
Published: October 2019

Hypoxia to heart or brain is a primary cause of heart failure or stroke. Studies have shown hypoxia increases the beta-adrenergic receptors (βARs) phosphorylation and dysfunction (Cheong et. al., 2016). These observations provide evidence that βARs can directly be regulated by hypoxia but less is known about the underlying mechanisms. We postulated that hypoxia shifts the homeostasis between kinase and phosphatase driven mechanisms may underlie βAR dysfunction. β2AR HEK 293 cells were exposed to hypoxia (2% O
2
) and assessed the mechanisms underlying desensitization (G-protein coupled receptor kinases, GRKs) and resensitization (Protein phosphatase 2A, PP2A). Six hours of hypoxia treatment resulted in increase of βAR phosphorylation and GRK2 expression. Assessment of βAR phosphorylation in the plasma membrane and endosomal fractions surprisingly, showed marked increase in β2AR phosphorylation in the endosomal fraction. Furthermore, we also observed that receptor associated PP2A activity was inhibited in the endosomes following hypoxia with minimal changes of activity at the plasma membranes. Similarly subjecting normal mice to 20 hours of hypoxia resulted in significant cardiac dysfunction (% FS: Normoxia 38.83% vs. Hypoxia 32.38%, P=0.0055; % EF: Normoxia 69.71% vs. Hypoxia 60.76%,P=0.0105) and was associated with significant increase in β2AR phosphorylation associated with significant loss in βAR function as measured by G-protein coupling adenylyl cyclase activity. Given that β-blockers confer beneficial effects, we tested whether β-blocker (propranolol) would prevent βARs phosphorylation under hypoxia or normoxia. Consistently, β-blocker treatment in normoxia results in increased β2AR phosphorylation however, remarkably β-blocker treatment in hypoxia results in loss of β2AR phosphorylation, reduction in GRK2 expression and increase in βAR-associated PP2A. These studies show that agonist-independent hypoxia-driven β2AR dysfunction can be ameliorated by β-blockers and the underlying mechanisms for this expected findings will be discussed in the presentation. These findings have significant clinical implications as understanding these mechanisms could provide novel insights into the benefits provided by β-blockers.
Abstract 591: Improving Cardiovascular Health Through DNA Repair
Authors: Wu Colin G
Published: October 2019

Accumulated DNA damage and oxidative stress play a critical role in the etiology of cardiovascular diseases. Malfunctions of human DNA repair proteins can lead to increased myocardial infarctions, ischemic heart disease, and congestive heart failure. The FANCJ helicase and the BRCA1 tumor suppressor are essential proteins that coordinate the repair of interstrand DNA crosslinks and double-stranded breaks in human cells. Their activities are especially needed in heart muscle, which undergoes tremendous physiological and chemical stress. Our limited understanding of how FANCJ and BRCA1 target damaged DNA structures remains a major barrier to research progress. As an independent investigator, my primary focus is to assess the impact of oxidative DNA damage on cardiovascular health through systematic dissection of the DNA repair pathways maintained by FANCJ and BRCA1. A detailed understanding of these molecular mechanisms would provide new diagnostic and therapeutic strategies to detect and to treat ischemic heart disease and congestive heart failure. Here, we have examined the DNA binding properties of FANCJ and BRCA1 using fluorescence spectroscopy and biolayer interferometry. We have also measured the viabilities of human cardiomyocytes and probed their sensitivity to reactive oxygen species by automated cell counting and single-cell electrophoresis. The DNA binding results, which describe precisely how FANCJ and BRCA1 organize onto damaged DNA sites, are compared with the cell-based analysis to correlate their DNA interaction patterns with cardioprotection. Our future work will utilize this platform to predict the risks of FANCJ and BRCA1 single-nucleotide polymorphisms that still have unknown cardiovascular effects.
Abstract 592: Cardioprotection During Ischemia by Induced Coronary Collateral Growth
Authors: Jamaiyar Anurag , Juguilon Cody , Cumpston Devan , Wan Weiguo , Wang Tao , Wang Zhiyuan , Gadd James , Enrick Molly , Chinchilla Sofia , McCabe Caige , Pu Autumn Y , Chilian William , Yin Liya
Published: October 2019

Patients with poorly developed coronary collateral networks have a poorer prognosis after a cardiovascular event than those with well-developed collaterals. The outcome of patients with metabolic syndrome (MetS) was worse than the patient without MetS. Interestingly, coronary collateral growth is impaired in MetS. We hypothesize that coronary collaterals are critical for the cardiac protection in ischemic heart diseases (IHD) and induction of coronary collateral growth (CCG) might ameliorate the outcome of patients with MetS. We study the cardiac protection by coronary collaterals in IHD and the underlying mechanism of impaired CCG in the MetSin a mouse model of CCG. A pneumatic snare was implanted and situated around the LAD. After the mice recovery from the surgery, periodic inflation of the snare occludes the LAD; thus, producing repetitive ischemia (RI). Cardiac function was measure with echocardiography. CCG was measured by myocardial blood flow with contrast echocardiography and by collateral numbers with micro-CT or by vascular density with fluorescence scope. Our preliminary results showed that with occlusion of the LAD, there was no blood flow in the ischemic area. However, after stimulation of CCG by RI, the blood flow the ischemic area was compensated by the grown coronary collaterals and cardiac function was reserved during ischemia. Diabetic mice failed to grow coronary collaterals after RI stimulation and there was no cardiac protection from coronary collaterals during ischemia. Moreover, we studied the roles of growth differentiation factor 11 (GDF11) and miR21 in the regulation of CCG and cardiac protection in IHD. GDF11 was downregulated and miR21 was upregulated in the hearts of Zucker Obese Fatty (ZOF) rats during CCG. We used the Gdf 11 knockout mice and miR 21 knockout mice to study the underlying mechanism. GDF11 is highly expressed in myocardial blood vessels, which suggested the role of GDF11 in vascular growth. Impaired CCG of mice on high fat and high sugar was restored by miR-21 knockout. These data suggest that miR-21 is involved in the impairment of CCG in MetS.
Abstract 602: Mir-182-5p is a Conserved Downstream Effector of Tbx5 Involved in Heart Development and Arrhythmia in Zebrafish
Authors: Guzzolino Elena , Pellegrino Mario , Ahuja Neha , Garrity Deborah , D'Aurizio Romina , Groth Marco , Baumgart Mario , Hatcher Cathy , Mercatanti Alberto , Evangelista Monica , Ippolito Chiara , Tognoni Elisabetta , Fukuda Ryuichi , Lionetti Vincenzo , Pitto Letizia
Published: October 2019

Background:
TBX5 mutations cause Holt-Oram syndrome (HOS) characterized by upper limb and cardiac malformations, but can also contribute to early-onset of atrial fibrillation. Focusing on miRNAs involved in TBX5 regulatory circuits with a cardiac relevant role, we identified miR-182-5p, belonging to miR-183 cluster, found upregulated in Tbx5-depleted hearts of mouse and zebrafish embryos.

Methods:
To functionally analyse the miR-182-5p role in developing heart, miR-182-5p was dysregulated in zebrafish zygotes of Tg(Myl7:EGFP) and Tg(myl7:gCaMP) transgenic lines. To stably deregulate miR-182-5p in zebrafish heart we exploited the Gal4/UAS system to restrict the miR-182 expression into cardiac context. For physiological analyses we performed the mechanogram of cardiac contraction and electrocardiogram recording. To understand miR-182-5p downstream regulation, in silico analyses, followed by ddPCR/real-time quantifications on dissected zebrafish hearts and rescue experiments both in transient and stable miR-182-5p overexpressing zebrafish embryos were performed.

Results:
Depletion of Tbx5 from cardiomyocytes increased the expression of miR-182 cluster family that is controlled by Kruppel-like factor 4 (KLF4), a transcription factor repressed by Tbx5. Both transient and stable upregulation of miR-182 in zebrafish affect heart morphology, calcium handling and the onset of arrhythmia while its cardiac-specific downregulation decreases cardiac defects in zebrafish HOS hearts. Expression analyses on selected miR-182-5p putative targets revealed that several calcium channel proteins resulted downregulated in miR-182-5p overexpressing hearts. Transgenic zebrafish line stably overexpressing miR-182-5p in the heart manifested arrhythmia overtime with or without cardiac structural defects.

Conclusion:
We identified miR-182-5p as a potential suitable target to interfere in the circuit between upstream genetic abnormalities and downstream effectors leading to arrhythmia occurrence.
Abstract 612: Automated Analysis of Displacement From Intravital Multiphoton Microscopy in Mouse Ventricle
Authors: Allan-Rahill Nathaniel H , Small David M , Lamont Michael R , Djakpa Salomon , Jhala Marvarakumari G , Zhu Yvette , Nishimura Nozomi
Published: October 2019

Background:
Multiphoton microscopy (MPM) has enabled
in vivo
time-lapse imaging of the heart that shows motion of cells within the tissue with micrometer resolution. We developed automated analysis techniques to quantify cellular motion from in vivo cardiac MPM images throughout the cardiac cycle.

Methods:
Intravital cardiac MPM of the beating mouse heart was performed on 26 week-old, C57Bl6 mice (n=6). Image volumes (100 μm deep) were acquired at 30 frames per second while recording the electrocardiogram and respiratory pressure. An image volume was reconstructed by assembling lines acquired nearest to a specified point in the cardio-respiratory phase space (Fig. a). Motion was calculated as the three-dimensional transformation required to register the reconstructed images to the image at the most stable cardiac phase.

Results:
Volumes were reconstructed in 50 intervals across the cardiac phase that show vasculature (intravenous Texas-red dextran, red) and cardiomyocytes (rhodamine 6G, cyan) moving across the field of view (Fig. b). Automated analysis indicated a maximum displacement occurring at 16 % (anterior-posterior), 36 % (base-apex) and 38 % (epi-endocardial) of the cardiac cycle defined by R-wave. Comparison by manual tracking of features across the cardiac cycle at a subset of phases (10 cardiac phases at peak exhalation) validated the automated measurement (Fig. c). Automated motion tracking shows superior performance in the spatial resolution and speed of analysis.

Conclusions:
We have shown a novel, fast, and accurate technique for characterizing cardiac motion from
in vivo
cardiac MPM to study the performance of the contractile cells in health and disease.
Abstract 622: Load-Independent Systolic Dysfunction and Altered Z-Disc Protein Phosphorylation in Swine with Stretch-Induced Myocardial Stunning
Authors: Weil Brian R , Rasam Sailee , Konecny Filip , Smith Cody , Qu Jun , Canty John M
Published: October 2019

Objective:
We recently demonstrated that a transient rise in left ventricular (LV) preload leads to a reversible reduction in LV ejection fraction in the absence of ischemia (“stretch-induced stunning”). The present study was designed to determine whether this phenomenon is characterized by impaired load independent LV systolic function and alterations in sarcomeric protein phosphorylation.

Methods:
Swine (n=9) received a 1 hour infusion of phenylephrine (PE; 18 mg/hour iv) to transiently elevate LV end-diastolic pressure (EDP). Load independent systolic function was derived from LV pressure volume loops collected during inferior vena cava occlusion at baseline and 30 min after PE (Transonic SciSense). In a subset of animals (n=5), LV tissue was collected 1 hour after PE for TMT-labeled, LC-MS-based quantitative phosphoproteomics using Orbitrap.

Results:
PE elicited a rise in LV EDP (9±2 to 29±3 mmHg, p<0.01), followed by a reduction in LV ejection fraction and LV dP/dt
max
30 min later. At this time, load independent indices of LV systolic function tended to exhibit a reduced slope with a significant rightward shift of the volume-axis intercept (V
0
), indicative of decreased contractility (
Table
). This was accompanied by altered phosphorylation of 16 sarcomeric Z disc proteins at 27 phosphosites, including increased phosphorylation of α actinin, α crystallin B chain, desmin, and myotilin.

Conclusions:
Stretch-induced stunning following transient preload elevation is characterized by load independent systolic dysfunction. Phosphoproteomics implicate alterations in Z disc protein phosphorylation as a mechanism underlying contractile dysfunction in this condition.
Abstract 645: CUBIC as an Alternative “Clearing” Method for Creating Hydrogel-based Structure in Organs
Authors: Tallam Meghana , Yuan Qize , Greer Meg , Kay Matthew W
Published: October 2019

Cardiac nerves have a major influence on cardiac health, heart failure, and sudden cardiac death. Understanding the impact of the cardiac nervous system requires in depth cell-specific analysis of the heart, which can be done using tissue clearing followed by 3D microscopic imaging. “Clearing” is the creation of hydrogel-based structures of organs where the lipids are removed without damaging the proteins of the organ. We are interested in using clearing to analyze cardiac neural anatomic pathways with high accuracy. Recently the CUBIC clearing method has gained momentum since it can clear tissue quicker than other methods. While previous research has shown qualitative images and descriptions to describe CUBIC efficacy, quantification of the clearing performance is lacking. The purpose of this study was to compare sequential images of cardiac tissue immersed in CUBIC solution alongside tissue absorbance spectra to measure the efficiency of the CUBIC method. To visualize cardiac neurons, we selectively expressed a fluorescent probe: an enhanced yellow fluorescent protein (EYFP) conjugated with the light-activated channel (channelrhodopsin (ChR2). Mice were crossbred to express EYFP+ChR2 in parasympathetic cardiac neurons which was driven by a choline acetyltransferase (ChAT) promoter using the Cre-Lox system. Hearts from these mice were excised and perfused with PFA as a fixative and perfused with the CUBIC solution. The tissue then was immersed in CUBIC solution and kept in an insulated shaker at 37°C. Each day, the tissue holder that held the cardiac tissue was taken out of the CUBIC solution and a white light from an LED was directed through the heart and the light intensity was recorded by a spectrometer. Beer-Lambert’s Law related light intensity and transmittance to absorbance to measure daily changes in the absorbance spectrum. Finally, we imaged the right atria of a cleared heart to capture the parasympathetic neural anatomic pathways using 2-photon confocal microscopy. We found that, that over the course of 14 days, the absorbance spectrum of the heart sample decreased over time, with the greatest reduction occurring between days 1 and 4. Future work would include decreasing environmental factors when measuring light intensity.
Abstract 655: Protein Arginine Methyltransferase 6 Controls Cardiac Hypertrophy by Differential Arginine and Lysine Methylation of Histone H3
Authors: Raveendran Vineesh , Al-Haffar Kamar , Kunhi Muhammed , Behlai K , Al-Habib Waleed , Eyjolsson Atli , Al-Buraiki Jehad , Poizat Coralie I
Published: October 2019

Heart failure remains a common cause of hospitalization and death worldwide. Heart failure can be caused by dysregulation of gene expression following abnormal expression of histone modifying enzymes. While lysine acetylation and methylation of histones have been the topic of many investigations, the role of arginine methylation in H3 has been overlooked. In an effort to understand regulatory mechanisms implicated in cardiac hypertrophy and heart failure, we assessed protein arginine methyl transferase (PRMT) members in the left ventricle of failing human hearts and control hearts. Our results show a specific up-regulation of PRMT6 mRNA in failing human hearts (5.95±2.16 fold in failing versus control, p=0.003) which also occurs in the compensatory phase of cardiac hypertrophy in mouse hearts subjected to pressure overload hypertrophy, and in neonatal rat ventricular myocytes (NRVM) stimulated with the hypertrophic agonist phenylephrine (PE). These changes are associated with a significant increase in arginine 2 asymmetric methylation of H3 (H3R2me2a) and reduced lysine 3 tri-methylation of H3 (H3K4me3) both in NRVM and
in vivo
. Importantly, forced expression of PRMT6 in NRVM stimulated with PE, enhances the expression of atrial natriuretic peptide (ANP). Conversely, silencing of PRMT6 reduces ANP protein expression and cell size, indicating that PRMT6 is critical for PE-mediated cardiac hypertrophy of NRVM. Also, silencing of PRMT6 reduces H3R2me2a, a mark associated with transcriptional repression. To evaluate the role of PRMT6 on cardiac contractility and global ion channel activity, we assessed contractility and global field potentials in live NRVM expressing normal and low level PRMT6 using the RTCA CardioECR system (ACEA Bioscience Inc.). Strikingly, reduced expression of PRMT6 drastically inhibits the contraction rate of NRVM, which is paralleled by a slight increase in the QT interval. All together, our results indicate that PRMT6 is a critical regulator of cardiac hypertrophy, implicating H3R2me2a as an important histone modification. Future studies investigating the specific gene programs regulated by PRMT6 are on their way. This study may help identify novel points of control to design new drugs for the treatment of heart failure.
Abstract 656: Construction and Application of an Epigenetic Atlas of the Human Heart
Authors: Prasher Dimple , Zhang Hao , McCarthy Patrick M , Oudit Gavin , Fedak Paul W , Greenway Steven C
Published: October 2019

Epigenetic modifications, including DNA methylation, regulate gene expression and contribute to the differentiation of cells. Tissues are characterized by methylation patterns that reflect their specific functions and origin. Currently, there is limited knowledge of the epigenetic patterns of the heart. To address this gap, we generated methylation profiles for all anatomical regions of the human heart. We hypothesize that unique differentially methylated regions (DMRs) of DNA exist for each cardiac region and that these patterns are disrupted in heart failure.

Using non-diseased cardiac tissue and reduced representation bisulfite sequencing on a Illumina platform, we generated genome-wide methylomes for the human right atrium (n=4), left atrium (n=4), right ventricle (n=4), left ventricle (n=4), aorta (n=3), pulmonary artery (n=2), mitral valve (n=3), tricuspid valve (n=3), aortic valve (n=3) and pulmonary valve (n=3). DMRs, defined as regions with significantly different mean methylation differences, were identified using Metilene.

For each tissue we identified between 10-20 million reads covering 8-10 million CpG methylation sites and 4-228 tissue-specific DMRs. There were relatively few (4) different DMRs between the left and right ventricles but 228 unique DMRs were found in the vessels (aorta and pulmonary artery) compared to the ventricles including regions upstream of
BMP3
and
FOXC1
, genes implicated in cardiogenesis. We then applied this approach and normal data to the analysis of disease. We generated additional left ventricular methylomes from adult (n=3) and pediatric (n=3) patients with heart failure. We identified 19 unique DMRs in the adult group and 107 in the pediatric group. Genes associated with pediatric HF DMRs included
ROCK1
and
FBLN2
that have been previously implicated in contraction and remodelling.

We have created an atlas of the human heart based upon differences in DNA methylation between the anatomical regions of the human heart. This data will help increase our understanding of cardiac development, identify new disease biomarkers and have application to a wide range of cardiac diseases
Abstract 705: Synergistic Activation of the Cardiac Enhancer Landscape During Cardiac Reprogramming
Authors: Garry Glynnis A , Hashimoto Hisayuki , Wang Zhaoning , Zhou Huanyu , Liu Ning , Bassel-Duby Rhonda , Olson Eric N
Published: October 2019

Direct cardiac reprogramming of fibroblasts to cardiomyocytes is an attractive therapeutic strategy to restore cardiac function following injury. The cardiac transcription factors Gata4, Mef2c, and Tbx5 are sufficient to directly reprogram fibroblasts to a cardiac fate and their cardiogenic activity is enhanced by the addition of Hand2 and Akt1. However, the mechanisms by which these transcription factors orchestrate this cell fate conversion remains elusive. To understand the mechanistic basis of cardiac reprogramming, we performed a genome-wide analysis of cardiogenic transcription factor binding sites and enhancer activation throughout cardiac reprogramming. We found that cardiogenic transcription factors act cooperatively by co-occupying regulatory elements enriched with Mef2 binding sites during cardiac reprogramming. Importantly, these transcription factors when overexpressed in isolation are incapable of activating reprogramming enhancer elements. Further, we discovered that the early reprogramming enhancer landscape most closely resembles that of the neonatal heart. Acquisition of enhancers associated with cardiac maturation occurred at later stages in reprogramming, with addition of Hand2 and Akt1 augmenting activation of this more mature enhancer landscape. Additionally, we constructed a cardiac reprogramming gene regulatory network which demonstrated downregulation of the EGF receptor signaling pathway. We found that inhibition of EGF receptor signaling augments reprogramming toward the cardiac phenotype. Our findings demonstrate that cardiac reprogramming is coordinated by synergistic transcriptional activation across a broad landscape of cardiac enhancers and define the epigenetic landscape of the cardiac phenotype.
Abstract 711: Therapeutic Effects of Engineered Human Pluripotent Stem Cell-derived Lymphatic Endothelial Cells on Experimental Lymphedema
Authors: Lee Shin-Jeong , Sohn Young-Doug , Sung Eun-Ah , Kim JungWoo , Yoon Young-sup
Published: October 2019

Current systems generating lymphatic endothelial cell (LEC) from human pluripotent stem cells (hPSCs) have limited value due to low purity, the use of undefined components for differentiation, and poor cell survival
in vivo
. Here, we developed a fully defined system to differentiate hPSCs into LECs and evaluated their therapeutic and engraftment potential when encapsulated in a nanomatrix gel. hPSCs were cultured with GSK3-β inhibitor for 3 days to induce differentiation into the mesodermal lineage. The mesodermal cells were cultured for another 6 days and double-sorted by PDPN and FLT4. These hPSC-PDPN
+
FLT4
+
cells showed highly purified and fully functional LEC characteristics
in vitro.
These hPSC-LECs express LEC markers such as PDPN, LYVE1, PROX1, and FLT4 at the mRNA level and the protein level, and formed tube-like structures in Matrigel. We next determined the lymphatic vascular reparative effects of engineered hPSC-LECs. After inducing lymphedema in the tail of mouse, hPSC-LECs, hPSC-LECs/PA-RGDS, human dermal lymphatic endothelial cells (hdLEC), PA-RGDS, or PBS were injected into the tail of mouse. Tail thickness significantly decreased in the groups injected with hPSC-LECs with or without PA-RGDS compared to the other groups at day 28. At day 45, mice injected with PA-RGDS encapsulated hPSC-LECs showed significant decrease in the tail diameter compared to all other groups including those injected with hPSC-LECs. This study demonstrated for the first time that PA-RGDS encapsulation can substantially improve lymphedema repair in mouse tail through enhancement of cell survival and lymphatic neovascularizationThis engineered hPSC-LEC therapy represents a novel option for treating lymphedema.
Abstract 719:
CRISPR/Cas9-
Based Knockout of the
TLR4
gene Enhances Secretion of Extracellular Vesicles With Anti-Inflammatory Properties From Human Cardiac Mesenchymal Stromal Cells
Authors: Schary Yeshai , Naftali-Shani Nili , Leor Jonathan
Published: October 2019

Background and Aim:
The environment of the failing and infarcted myocardium drives resident and transplanted mesenchymal stromal cells (MSCs) toward a pro-inflammatory phenotype and restricts their survival and reparative effects in a mechanism mediated by the toll-like receptor 4 (
TLR4
). CRISPR is a promising tool for genome editing of DNA in cells, which raises hope for therapeutic genome editing in the clinic. We hypothesize that
ex-vivo
knockout (KO) of the human
TLR4
gene by CRISPR would switch human cardiac MSCs (hMSCs) to an anti-inflammatory, reparative phenotype that could prevent remodeling of the left ventricle after myocardial infarction (Fig. 1A).

Methods and Results:
For gene editing, we electroporated a Cas9 nucleoprotein aimed to KO the
TLR4
gene. To assess the inflammatory response, we analyzed cell secretome. We achieved up to a 68% (out of 400,000 cells) success rate in editing the genome of hMSCs (

R
2
=0.93

). The
TLR4
KO hMSCs secreted smaller extracellular vesicles (sEVs) compared with unedited hMScs (Fig. 1B
p<0.001
) and decreased the secretion of most pro-inflammatory (e.g. IL-1α) and pro-fibrotic (e.g. IL-10) cytokines from edited compared with unedited hMSCs (Fig. 1C, D). Additionally, sEVs from
TLR4
KO cells stimulated hMSC migration by “wound healing” scratch assay (
p
<
0.001
).

Conclusion:
Our preliminary results show, for the first time, that CRISPR-based KO of the human
TLR4
gene in hMSCs inhibits inflammatory cytokine secretion and facilitates a reparative response by human-cardiac MSCs in vitro. The precise and efficient ex vivo gene editing could provide a newly engineered cell line to improve the outcome of hMSC-based cell therapy.
Abstract 722: Unveiling Ccbe1 Role as a Modulator of Cardiomyocyte Differentiation
Authors: Silva Marta M , Vicente Pedro , Simão Daniel , Terrasso Ana P , Inácio José M , Belo José A , Gomes-Alves Patrícia , Serra Margarida , Alves Paula M
Published: October 2019

Chronic heart failure is a major unmet clinical need arising from the loss of viable and functional cardiac muscle, representing a major cause of mortality worldwide. There is a need to identify key molecules and signaling pathways acting on the coronary vasculature system towards regenerative therapies. Human induced pluripotent stem cells (hiPSC) are an attractive cell source to understand the regulatory networks involved in cardiac commitment and cardiomyocyte (CM) differentiation. Particularly, CCBE1 (collagen and calcium-EGF biding domain 1) has been studied as a secreted protein critical for lymphatic/cardiac vascular development and recent reports have proposed that CCBE1 may potentially be used to restore cardiac tissue upon heart injury, through CCBE1-mediated cardiac commitment and/or augmentation of lymphangiogenesis. Therefore, the goal of our work is to understand the molecular pathways underlying CCBE1-based cardiac commitment in loss-of-function studies. By exploring gene editing and “omics” tools we aim to unveil the molecular basis of CCBE1-induced cardiogenesis and provide novel insights towards the development of CCBE1-mediated therapeutic strategies for cardiac regenerative medicine. To selectively knock-down (KD) CCBE1 expression along hiPSC cardiac differentiation, we used modified hiPSC line with CRISPR interference technology (CRISPRi-hiPSC). The CCBE1 KD led to a reduction on the expression of cardiac troponin marker
TNNT2
and on the ratios of MYH7:MYH6 and TNNI3:TNNI1. The CCBE1 KD-derived CMs also presented an immature-related ultrastructure, suggesting that CCBE1 may modulate the CM phenotype. On the other hand, the EC differentiation was not impaired by CCBE1 KD. A comprehensive and integrated characterization of the transcriptome and proteome along the differentiation in the presence or absence of CCBE1 is being pursued to identify the key players at cardiac mesoderm and cardiac progenitors stages and their interactions with CCBE1. This work opens new avenues for the identification of CCBE1-modulated proteins/pathways in cardiac commitment, which may contribute for novel cell-based or cell-free approaches towards more efficacious cardiac regenerative therapies.
Abstract 723: Cortical Bone Derived Stem Cells Modulates T Cell Response After Myocardial Injury
Authors: Wagner Marcus J , Ma Lena , Bryan Christopher F , Vyas Ritu S , Kurian Justin J , Khan Mohsin , Mohsin Sadia
Published: October 2019

Rationale:
The ischemic environment following myocardial infarction induces a harsh, pro-inflammatory state that facilitates the removal of necrotic tissue. Manipulation of T cell responses after cardiac injury directly impacts the outcome of the reparative response. Previous studies have demonstrated Cortical Bone Derived Stem Cells (CBSCs) improve cardiac function after myocardial infarction. CBSCs produce a diverse paracrine profile; whether this can lead to the modulation of T cell response by manipulating T cell subsets and allowing for increased cardiac wound healing post-MI remains unknown.

Objective:
To determine whether CBSCs secrete a paracrine profile that promotes wound healing by modulating T cell response following myocardial injury.

Methods and Results:
CD3+ T-lymphocytes were isolated from the spleen of C57BL/6J male mice ages 8 to 12 weeks via Fluorescent Activated Cell Sorting (FACs). CBSC secretome was introduced to T cell populations via a transwell culture system in the presence of CD3/28 coated beads with IL2. T-regulatory (Treg) populations were quantified via FACs 24 hours after
in vitro
CBSC exposure. Treg populations exposed to CBSC secretome in hypoxic (1% O2) conditions expanded to 26.9% of the CD4+ compartment, a 4.5 fold increase compared to control conditions. This result was further confirmed via qRT-PCR analysis. T cell culture secretome was quantified via proteome profiling of 111 soluble cytokine proteins 24 hours post CBSC exposure. CBSCs express a robust chemotaxis signature that promotes immune cell recruitment. T cells cultured in the presence of CBSC (pre-conditioned or co-culture conditions) exhibit increased CCL5, CCL22, CD40, CXCL1, and Lipocalin-2. C-reactive protein, a pro-inflammatory marker, was decreased by ~5.5 fold in the presence of CBSCs, promoting wound healing.

Conclusions:
CBSCs secrete a diverse paracrine profile that promotes Treg expansion during hypoxic conditions. Expansion of Treg populations post-CBSC exposure in the post-IR heart leads to increased inflammation resolution and improved myocardial wound healing, allowing for increased cardiac function and repressed cardiac remodeling.
Abstract 724: Neonatal Heart Regeneration Preserves Native Ventricular Biomechanical Properties After Myocardial Infarction
Authors: Wang Hanjay , Bennett-Kennett Ross , Paulsen Michael J , Hironaka Camille E , Thakore Akshara D , Farry Justin M , Eskandari Anahita , Lucian Haley J , Wu Matthew A , Imbrie-Moore Annabel , Steele Amanda N , Stapleton Lyndsay M , Dauskardt Reinhold H , Woo Y. Joseph
Published: October 2019

Introduction:
Neonatal mice exhibit natural heart regeneration after myocardial infarction (MI) until postnatal day 7 (P7). Whether this regenerated muscle is similar biomechanically to native myocardium remains unknown. We hypothesized that neonatal heart regeneration preserves native left ventricular (LV) biomechanical properties after MI.

Methods:
C57BL/6J mice (n=58) underwent sham surgery or left anterior descending coronary artery ligation on postnatal day 1 (P1) or P7. After 4 weeks, echocardiography was performed. The explanted anterolateral LV wall was mounted for lenticular hydrostatic deformation testing (Fig 1A-B) and pressurized to 130 mmHg with 37°C saline to impart a homogeneous stress load. Surface strain was tracked to calculate a multiaxial composite tissue modulus. Data are expressed as mean±SEM.

Results:
P1 MI and sham mice (n=14, each) had similar post-MI LV wall thickness (0.61±0.05 cm vs 0.66±0.03 cm, p=0.39), end-diastolic diameter (3.14±0.10 cm vs 3.05±0.12 cm, p=0.57), and ejection fraction (58.2±4.1% vs 55.2±4.4%, p=0.63). P7 MI mice (n=14) had significant LV wall thinning (0.40±0.04 cm vs 0.71±0.02 cm, p<0.0001), end-diastolic LV enlargement (4.24±0.16 cm vs 3.34±0.09 cm, p<0.0001), and depressed ejection fraction (29.7±4.9% vs 61.7±4.0%, p<0.0001) compared to P7 shams (n=16). While LV tissue modulus for P1 MI and sham mice were similar (460.9±46.4 kPa vs 527.1±61.2 kPa, p=0.40, Fig 1C), the modulus for P7 MI mice was significantly elevated compared to that for P7 shams (1099.6±175.1 kPa vs 616.0±95.2 kPa, p=0.02, Fig 1D).

Conclusion:
In a neonatal mouse MI model, regenerated LV muscle has similar biomechanical properties as native LV muscle.