bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 54: MicroRNA Let-7d-3p in Heart Failure
Authors: Wong Lee Lee , Saw Eng Leng , Zhou Yue , Wang Peipei , Richards Arthur M
Published: June 2021

Circulating microRNA (miRNAs) levels are dysregulated in heart failure (HF) suggesting possible diagnostic and prognostic applications. In addition, miRNAs may yield insights into the pathophysiology of HF and provide targets for therapeutic intervention. We sought to identify the diagnostic utility of miRNAs in HF and to further investigate selected miRNAs as potential targets for therapeutic intervention. We hypothesized that distinct dysregulated circulating miRNAs in HF may be potential therapeutic targets to improve outcomes for HF.

Methods:
Plasma from 338 well-characterized HF patients and 208 age-matched controls were collected for qPCR based miRNA profiling. Selected miRNAs of interest were further investigated using myocyte cell line, H9C2 and primary neonatal rat ventricular cardiomyocytes, NRVM.

Results:
Seventy-four miRNAs (AUC>0.65, p<0.001) were significantly dysregulated for discrimination of HF from non-HF controls on receiver-operator analysis. Seven miRNAs of interest were selected for further investigation. Of these, let-7d-3p had the most up regulated miRNA fold change=1.32 (p<0.001 after FDR). Functional screening using mimic transfection demonstrated that let-7d-3p mimic increased CCK-8 activity and decreased LDH release in both H9C2 and NRVM during hypoxia. By in silico target prediction (miRDB, microRNA.org and Targetscan) and qPCR, mRNA targets YY1, KLF9, KLF12 and MEX3C were identified as candidate targets for let-7d-3p and were significantly downregulated by let-7d-3p mimic. This suggests that let-7d-3p may play a role in signaling pathways pertinent to cardiac development (YY1), cell proliferation/survival (KLF9 and KLF12) and adiposity (Mex3c).

Conclusions:
We identified miRNAs that were dysregulated in clinical HF and our preliminary data on let-7d-3p suggests roles in the pathophysiology of HF that warrant further investigation.
Abstract 55: The Natural Course of Myocardial Infarction in Large Animal Models; a Systematic Review and Meta-analysis
Authors: Zwetsloot Peter-Paul , Kouwenberg Lisanne E , Sena Emily S , Eding Joep E , Sluijter Joost P , Pasterkamp Gerard , Doevendans Pieter A , Chamuleau Steven A , van Hout Geert P , Jansen of Lorkeers Sanne J
Published: June 2021

Background:
Large animal models of myocardial infarction (MI) are essential for the development of novel therapeutic strategies. However, it is unclear which experimental factors are independent determinants of disease progression outcomes and the fidelity with which these models resemble MI in humans. The aim of this study was to investigate systematically what independently influences the outcome after MI in large animal models and determine which methodological factors influence primary endpoints and disease progression.

Methods:
We used control animal data from two meta-analyses of large animal models of MI. We performed univariable and multivariable meta-regression to analyze if potential relevant variables were influencing outcomes; infarct size as a ratio of the area at risk (IS/AAR), as a ratio of the left ventricle (IS/LV) and ejection fraction (EF). Pre-defined independent variables were species, sex, weight, ischemia model (open vs closed), occlusion type (temporary vs permanent), occluded vessel, follow-up duration, use of co-medication, use of immunosuppression and study quality. Outcomes and variables were complemented per dataset if needed.

Results:
Our analyses yielded 246 relevant studies, reporting 1500, 1221 and 775 control animals for IS/AAR, IS/LV and EF respectively. Multivariable meta-regression showed IS/AAR was influenced by species (p<0.001), sex (p=0.03), co-medication (p=0.01), occlusion type (p<0.001), occluded vessel (p=0.002) and follow-up (p=0.001). For IS/LV occlusion type (p=0.03), occluded vessel (p=0.03) and study quality (p=0.03) showed significant effects. EF measurements revealed that species (p=0.04), sex (p=0.04) and occluded vessel (p=0.05) were independent predictors. Using these variables, we can partially predict these outcomes for certain study setups.

Conclusion:
Many methodological variations exist in the design of large animal MI studies. This should be taken into account when selecting a model to study therapy efficacy. We provide evidence that disease manifestation and progression greatly depend on certain biological characteristics, e.g. location of MI and sex of the patient. It is therefore possible that therapies have a different effect in specific patient populations.
Abstract 63: Reduction in Glucocorticoid Receptor Expression Attenuates Pressure Overload-induced Cardiac Hypertrophy and Promotes Heart Failure in Mice
Authors: Wu Rongxue , Knapp Maura , Zheng Mei , Liao James K
Published: June 2021

Background:
Left ventricular hypertrophy (LVH) is an independent risk factor for heart failure and sudden death. In addition, LVH is also a compensatory mechanism that helps the heart cope with pressure overload. Stress is considered one factor that is related to cardiac outcomes. Glucocorticoids are primary stress hormones, whose role in the heart is poorly understood. Here, we hypothesize that a reduction in the expression of the glucocorticoid receptor (GR) would decrease cardiac hypertrophy in response to pressure overload.

Methods and Results:
The GR homozygous mutation (GR-/-) is embryonic lethal. However, GR heterozygous mice (GR+/-) show a normal phenotype. We subjected GR+/- mice to transverse aortic constriction (TAC). At four weeks after TAC, the ratio of heart weight to tibia length increased significantly in wild-type mice (control) littermates compared with GR+/- mice. Cardiac myocyte size was also smaller in GR+/- mice vs controls, suggesting an attenuated cardiac growth response in these mice. In addition, GR+/- hearts displayed increased cell death and enhanced fibrosis in response to TAC. Cardiac function, determined by EF% and FS% (measured using the Vevo2100 imaging system), was significantly reduced in GR+/- mice compared with controls at eight weeks post-operation, while LVEDD was increased. Together, with the increased ratio of lung weight to body weight in GR+/- mice at eight weeks following TAC, this suggests an exaggerated heart failure in GR+/- mice. In vitro, hydrocortisone-induced cell growth in H9c2 cells was abolished by GR knockdown using siRNA. Finally, we looked at the mechanisms by which GR may play a role in the development of hypertrophy. We found reduced ERK-JNK activity in GR+/- hearts, suggesting that the reduced hypertrophic response in GR+/- mice occurs, at least partially, through abolished JNK and ERK activity.

Conclusion:
The glucocorticoid receptor is required for cardiac hypertrophy and protects the heart from heart failure during cardiac pressure overload.
Abstract 65: Latrophilin-2 is a Specific Cell-surface Marker for Cardiac Progenitor Cells and Specifies Cardiac Lineage Commitment and Development
Authors: Cho Hyun-Jai , Lee Choon-Soo , Lee Jin-Woo , Han Jung-Kyu , Yang Han-Mo , Kwon Yoo-Wook , Kim Hyo-Soo
Published: June 2021

Backgrounds:
The identification of a lineage-specific marker plays a pivotal role in understanding developmental process and is utilized to isolate a certain cell type with high purity for the therapeutic purpose. We here report a new cardiac-specific marker, and demonstrate its functional significance in the cardiac development.

Methods and Results:
When mouse pluripotent stem cells (ES and iPS cells) were stimulated with BMP4, Activin A, bFGF and VEGF, they differentiated into cardiac cells. To screen cell-surface expressing molecules on cardiac progenitor cells compared to undifferentiated mouse iPS and ES cells, we isolated Flk1+/PDGFRa+ cells at differentiation day 4 and performed microarray analysis. Among candidates, we identified a new G protein-coupled receptor, Latrophilin-2 (LPHN2) whose signaling pathway and its effect on cardiac differentiation is unknown. In sorting experiments under cardiac differentiation condition, LPHN2+ cells derived from pluripotent stem cells strongly expressed cardiac-related genes (Mesp1, Nkx2.5, aMHC and cTnT) and exclusively gave rise to beating cardiomyocytes, as compared with LPHN2- cells. LPHN2-/- mice revealed embryonically lethal and huge defects in cardiac development. Interestingly, LPHN2+/- heterozygotes were alive and fertile. For the purpose of cardiac regeneration, we transplanted iPS-derived LPHN2+ cells into the infarcted heart of adult mice. LPHN2+ cells differentiated into cardiomyocytes, and systolic function of left ventricle was improved and infarct size was reduced. We confirmed LPHN2 expression on human iPS and ES cell-derived cardiac progenitor cells and human heart.

Conclusions:
We demonstrate that LPHN2 is a functionally significant and cell-surface expressing marker for both mouse and human cardiac progenitor and cardiomyocytes. Our findings provide a valuable tool for isolating cardiac lineage cells from pluripotent stem cells and an insight into cardiac development and regeneration.
Abstract 66: Ascorbate Prevents Hypertension, Proteinuria and Concentric Hypertrophy by Balancing the Nitroso-redox System in a Model of Preeclampsia, the S-nitrosoglutathione Reductase (gsnor) Deficient Mice
Authors: Kulandavelu Shathiyah , Dulce Raul A , Takeuchi Rosemeire K , Balkan Wayne , Hare Joshua M
Published: June 2021

Introduction:
Preeclampsia (PE), a leading cause of maternal and fetal mortality and morbidity, is characterized by increased levels of reactive oxygen species (ROS) and S-nitrosylated protein, and decreased levels of the antioxidant, ascorbate (Asc). Mice lacking S-nitrosoglutathione reductase (GSNOR
–/–
), a denitrosylase that regulates protein S-nitrosylation, exhibit a PE-like phenotype including maternal hypertension, proteinuria, cardiac concentric hypertrophy and impaired placental vascularization. We hypothesize that the PE-like phenotype is mediated by nitroso-redox imbalance and nitrosative stress and can be rescued with ascorbate treatment.

Methods:
Pregnant GSNOR
–/–
and control (WT) mice (n=5-7) were provided drinking water ± Asc beginning at day 0.5 of gestation (E0.5). We determined blood pressure using a Millar catheter, relative wall thickness (RWT) by echocardiography, and placental vascularization by isolectin staining. Cardiomyocytes (CM) were isolated at late stage pregnancy (E17.5) and fluorescent dyes used to determine levels of ROS (2’7’-dichlorofluorescein), nitric oxide (NO, diaminofluorescin) and peroxynitrite (dihydrorhodamine 123).

Results:
Isolated CMs from pregnant GSNOR
–/–
hearts, exhibited elevated levels of ROS (2.48±0.39 vs. 1.58±0.18 ΔF/F
0
), free NO (6.65±0.43 vs. 5.59±0.26 ΔF/F
0
) and peroxynitrite (0.75±0.04 vs. 0.39±0.03 ΔF/F
0
) compared to WT. These increases were prevented with Asc treatment (P<0.01), which completely rescued the PE phenotype in GSNOR
–/–
mothers, including hypertension (105±2 mmHg vs. 95± mmHg in Asc-treated, P<0.05), proteinuria (P<0.05) and RWT (0.56±0.04 vs. 0.45±0.03 in Asc-treated (P<0.05). Placental vascularization was also significantly improved with Asc treatment in GSNOR
–/–
mothers. Asc had no significant effect in WT mice.

Conclusion:
Our findings indicate that nitroso-redox imbalance and nitrosative stress contributes to PE in mice. Asc treatment balanced the nitroso-redox system and rescued the pathological phenotypes in GSNOR
–/–
mice, suggesting that it can be used therapeutically to treat or prevent preeclampsia.
Abstract 70: Characterization of Advanced Glycation End Products in a Myocardial Infarction Animal Model
Authors: Fracasso Bianca , Rangel Juliana O , Olsen Virgílio , Phaelante Amanda , Curuja Fernanda , Lopes Amanda , Rohde Luis E , Biolo Andreia , Andrades Michael
Published: June 2021

Advanced glycation end products (AGE) are molecules produced by oxidative and inflammatory metabolism. Studies have shown that increased AGE levels worse the prognosis of the cardiovascular diseases. However, there is no characterization of AGE generation in a representative animal model of myocardial infarction (MI). Thus, our goal was to characterize the AGE formation in plasma and cardiac tissue in an animal model of MI. Male Wistar rats (3 month-old) were divided in 2 groups: Sham (n = 15) and MI (n = 14) and followed by 12 week. MI was induced by left anterior descending coronary artery (LAD) permanent ligation and the animals were stratified in 2 groups according to ejection fraction (EF) median at 2 days post-MI. MI induced 47% and 29% increase in heart weight/final body weight ratio in MIlowEF MIhighEF group, respectively. There was no significant difference among the groups in the plasma levels of reactive free amines, fluorescents AGE, Nε-(Carboxymethyl)lysine (CML) and Nε-(Carboxyethyl)lysine (CEL) in a 12-week follow-up. However, a significant decrease in the levels of yellowish-brown colored AGEs was revealed in MIlowEF group when compared to Sham. In the cardiac tissue homogenate there was no difference in the amounts of reactive free amines and carbonyl protein among the groups. Our results suggest that a widely employed animal model of MI partially agrees with the human manifestation of the ischemic heart disease concerning the AGEs metabolism. Thus, its use must be employed with caution when studying AGEs signaling or anti-AGE drugs.
Abstract 75: Cardiomyocyte-specific Conditional Deletion of GSK-3β Leads to Global Metabolic Defects and Cardiac Dysfunction in a HFD Induced Obesity Model
Authors: Lal Hind , Ahmad Firdos , Verma Vipin K , Tumuluru Samvruta , Parikh Shan , Zhang Qinkun , Force Thomas L
Published: June 2021

Obesity has reached epidemic proportions worldwide and is associated with increased risk of cardiovascular and metabolic disease, resulting in enhanced morbidity and mortality. Indeed, the primary cause of mortality in patients with diabetes is cardiovascular disease, accounting for 50-80% of deaths. Numerous studies have implicated GSK-3β in the pathogenesis of insulin resistance, metabolic syndrome and diabetes. However, these studies are primarily relies on non-isoform specific inhibitors. We and others have reported not only isoform-specific functions of GSK-3 but also their distinct roles in various tissues. At present, all available inhibitors of GSK-3 are non-isoform-specific; therefore it’s impossible to assess the isoform-specific functions by employing these agents. The primary goal of present study was to determine the role of cardiac GSK-3β in obesity-induced metabolic perturbations and cardiac dysfunction. An oral glucose tolerance test (GTT) was performed on cardiomyocyte-specific GSK-3β KO (GSK-3β KO) and controls. At baseline, GSK-3β KO and controls displays a comparable lean mass, fat mass and oral glucose tolerance. To determine the functional role of cardiac GSK-3β in obesity-induced glucose intolerance, GSK-3β KO and controls were subjected to HFD for 24 wks and an oral GTT assay was performed on fasting mice (6h). The hyperglycemic response was significantly increased in the obese GSK-3β KO mice in comparison to obese WT mice. Importantly, body weight and food consumption were comparable between groups confirming that observed glucose intolerance in GSK-3β KO were not confounded by variable body mass composition. Furthermore, HFD leads to accelerated cardiac dysfunction in GSK-3β KO hearts compared to controls as reflected by significantly reduced ejection fraction (EF) and functional shortening. In summary, these data suggest that cardiac-specific deletion of GSK-3β lead to systemic glucose intolerance and cardiac dysfunction. These findings suggest that cardiac GSK-3β is required for the maintenance of global metabolic homeostasis and cardiac function in diabetic hearts and strategies to maintain GSK-3β activity may lead to therapeutic benefits for the disease.
Abstract 77: Metabolic Reprograming Driven by Interaction AMP-activated Protein Kinase and Coactivator SRC-2 Regulate Cardiac Stress Response
Authors: Nam Deok Hwa , Suh Ji Ho , Reineke Erin L
Published: June 2021

Flexibility in fuel source for the heart is necessary to meet ATP demands during a high and variable workload such as transaortic constriction (TAC). During such chronic metabolic and hemodynamic stresses, shifts occur in substrate utilization in the heart based on magnitude of the demand, oxygen and nutrient availability. These shifts directly affect the amount of ATP available for other stress responsive pathways and therefore their efficacy. Communication from metabolic changes to these other pathways is crucial. As a result, it is important understand the molecular communication between metabolic and hypertrophic pathways under stress. We have found that chronic loss of cardiomyocyte SRC-2 and transient activation of AMPK both effect metabolic inputs prior to TAC and result in a blunted hypertrophic response. We hypothesized that SRC-2 and AMPK are critical upstream regulators of the cardiac stress response controlling metabolic changes translating into altered hypertrophy. Furthermore, we proposed that they could be regulating each other to control both the temporal changes to the metabolic machinery through AMPK and long-term genetic changes through SRC-2. We identified AMPK as a coregulator of SRC-2 activity and confirmed a direct interaction between them in cardiac cells. We have identified putative AMPK phosphorylation sites on SRC-2 that control SRC-2 stability and activity. Conversely, we found by ChIP that SRC-2 localized to AMPK subunit genes. Additionally, we performed co-immunoprecipitation and luciferase reporters and found that SRC-2 can interact with AMPK subunits and regulate its activity. We believe that disruption of these interactions is a main determinant in efficient hypertrophy in response to metabolic changes. Our results suggest the basis of a mechanism whereby energetic changes are sensed via AMPK and translated to transcriptional machinery of diverse targets via SRC-2 which allows AMPK to effect multiple cardiac transcription pathways. This mechanism provides insights into the role of AMPK-SRC-2 interaction in regulating myocardial function, metabolism, hypertrophy, and the progression to heart failure.
Abstract 84: Remodeling of Repolarization and Arrhythmia Susceptibility in a Myosin Binding Protein C Knockout Mouse Model
Authors: Toib Amir , Mohsin Sadia , Zhang Xiaoxiao , Zhang Chen , Troupes Constantine D , Wallner Markus , Berretta Remus M , Sharp Thomas E , Borghetti Giulia , Trappanese Danielle M , Chen Xiongwen , Houser Steven R
Published: June 2021

Background:
Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease (1:500 in the general population) and amongst the leading causes of sudden cardiac death (SCD) in the young. There is lack of an in-depth understanding of cellular mechanisms leading to SCD in HCM. Prior studies that explored the cellular electrophysiological alterations in HCM, focused on the role of calcium and sodium currents in the development of cardiac hypertrophy and arrhythmia. Repolarizing potassium currents in HCM have not been investigated in detail. Better definition of the cellular substrates for arrhythmias could improve risk stratification and lead to potential novel therapeutic targets for prevention of SCD.

Objective:
To characterize the arrhythmogenic phenotype, cellular electrophysiological properties and repolarizing potassium currents in Myosin binding Protein C (MyBPC) knockout (KO) mice.

Methods and results:
MyBPC KO mice demonstrate overt phenotype with severe hypertrophy and cardiac dysfunction compared to wild type (WT) control mice, as evidenced by echocardiography and heart weight/body weight analysis. Telemetric electrocardiographic recordings reveal prolongation of the QTc interval in the KO compared to WT control mice (53.2 ± 7.4 vs 30.5 ± 5 msec, n=4 KO, 4 WT, p< 0.05) with frequent arrhythmias including preexcitation, ventricular and supraventricular tachycardia. Whole cell current and voltage clamp studies, comparing KO to WT mice, demonstrate myocyte hypertrophy (469 ± 44 vs 234 ± 15 pF, n=18 KO,15 WT, p<0.01), action potential prolongation (Action potential duration (APD)50 of 30.4± vs 17.7 msec, APD90 of 125.7± vs 74.7 msec, n=11 KO, 12 WT, p <0.05) and decreased repolarizing potassium currents (n=15 KO, 14 WT): I
k
Peak (-120 to 60 mV) decrease of 33-39%, I
k
1 (-120 to -60 mV) decrease of 16-30%, I
ks
(20 to 60 mV) decrease 21-23% and I
to
(0 to 60mV) decrease of 46-61%.

Conclusion:
MyBPC KO mice demonstrate remodeling of repolarizing potassium currents, action potential and QTc intervals prolongation that could account for the arrhythmia susceptibility.
Abstract 85: Interactions Between Cardiac Myosin Binding Protein C and Actin Contribute to the Regulation of Cardiac Contraction
Authors: van Dijk Sabine J , Bezold Kooiker Kristina , Harris Samantha P
Published: June 2021

Cardiac myosin binding protein C (cMyBP-C) is a regulatory muscle protein that is essential for proper cardiac contraction, and mutations in cMyBP-C are commonly associated with hypertrophic cardiomyopathy. cMyBP-C not only interacts with myosin, but with actin as well.
In vitro
studies have demonstrated that multiple functions of cMyBP-C could readily be explained by an interaction between cMyBP-C and actin, but the
in vivo
significance of cMyBP-C binding to either myosin or actin is not well understood. Here we created transgenic mice with a single point mutation (L348P) in a key binding domain of cMyBP-C that enhances the binding affinity of cMyBP-C for actin
in vitro
(Bezold
et al
, JBC 2013) to gain insights into the relevance of cMyBP-C binding to actin in working hearts. Echocardiograms from 3 month old male L348P-Tg mice (N=23) and non-transgenic (nTg, N=17) controls were used to assess systolic and diastolic function. Results showed significantly prolonged isovolumetric relaxation time (L348P-Tg: 18.5±0.6 vs nTg: 10.2±0.3 ms) and slower movement of the mitral valve annulus (E’: -17.2±1.5 vs -32.2±1.6 and A’: -9.1±1.7 vs -17.6±1.0 mm/s, p<0.05), accompanied by slower inflow of blood into the left ventricle (reduced E and A, prolonged mitral valve deceleration time). Pressure-volume measurements showed significantly reduced rates of pressure decay in L348P-Tg mice (Tau Glantz: 39.1±2.6 vs 12.7±0.9 ms) and an increased end-diastolic pressure volume relationship (0.13±0.02 vs 0.07±0.01). We challenged mice with acute beta-adrenergic stimulation (isoprenaline injection) to determine whether the L348P mutation affected contractile reserve. Isoprenaline had little effect on diastolic parameters, but revealed systolic dysfunction in L348P-Tg mice as evident from a blunted increase in contraction (e.g. fractional shortening after isoprenaline: 38.5±1.3 vs 44.2±1.7%). Taken together our results show for the first time that interactions between cMyBP-C and actin are relevant for functioning of the whole heart. Increasing the cMyBP-C-actin interaction by the L348P mutation caused increased stiffness of the left ventricle, slowed relaxation and diastolic dysfunction. Results also suggested that the L348P mutation reduces contractile reserve.
Abstract 86: Compromised Sarcolemmal Membrane Repair Contributes to the Progression of Heart Failure
Authors: Bhattacharya Sayak , Xu Zhaobin , Gushchina Liubov , Appikatla Prasanthi , Beck Eric X , Alloush Jenna , Kilic Ahmet , Mohler Peter J , Janssen Paul M , Weisleder Noah
Published: June 2021

A conserved sarcolemmal membrane repair response exist to counteract membrane damage and restore membrane barrier function in order to maintain normal cellular homeostasis. This response can involve various mechanisms including activation of signaling pathways that trigger vesicular trafficking to the site of injury followed by vesicular fusion with the damaged portion of the membrane to patch the membrane disruption. Previous studies indicate that compromised repair capacity can exacerbate cardiac injury while increasing membrane repair capacity can reduce cardiac pathology. In our studies, membrane repair assays on cardiac and non-cardiac cell lines demonstrated that this process is dependent on activation of the phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) signaling axis through the downstream target Akt1. One mechanism found to increase membrane repair following PI3K/Akt1 activation is elevated exocytotic and endocytotic activity. Further studies indicate that the PI3K/Akt1 pathway is relevant to membrane repair in native hearts. Thick slices of myocardium from explanted human and mouse hearts were probed using multi-photon microscopy to determine the membrane repair capacity. These studies indicate decreased repair capacity in failing human myocardium as well as in mouse hearts following transaortic constriction (TAC). This membrane repair response requires PI3K/Akt1 signaling as genetically modified mice null for Akt1 show compromised sarcolemmal membrane repair. Additionally, PI3K or Akt1 inhibition prevents membrane resealing in non-failing human or mouse myocardium. The compromised membrane repair observed in failing human myocardium can be ameliorated by PI3K or Akt1 agonists. Treatment of TAC mice with multiple therapeutic compounds known to increase membrane repair capacity can minimize the development of structural and functional hallmarks of heart failure. Our results indicate that failing cardiomyocytes exhibit compromised membrane repair and that increased PI3K/Akt1 signaling can increase repair capacity thereby demonstrating potential as a heart failure treatment.
Abstract 87: Endothelial Nogo-B Regulates Sphingolipid Biosynthesis to Promote the Onset of Pathological Hypertrophy During Chronic Pressure Overload
Authors: Zhang Yi , Huang Yan , Cantalupo Anna , Azevedo Paula S , Siragusa Mauro , Bielawski Jacek , Giordano Frank J , Lorenzo Annarita D
Published: June 2021

Chronic pressure overload leads to an initial compensatory cardiac hypertrophy, and eventually to heart failure. The mechanisms regulating the transition from adaptive to pathological cardiac hypertrophy remain elusive. We recently discovered that endothelial Nogo-B, a membrane protein of the ER, regulates vascular functions by inhibiting the rate-limiting enzyme in
de novo
sphingolipid biosynthesis, serine palmitoyltransferase (SPT). Here, we show that sphingolipids produced by the vasculature, particularly S1P, protect the heart function during pressure overload, through a paracrine mode of action. SPT activity is upregulated in banded hearts
in vivo
, as well as in TNF-α-activated endothelium
in vitro
, and loss of Nogo-mediated brake on SPT increases the production of S1P, which enhances the coronary vasculature compliance to high pressure and endothelial barrier. Hence, mice lacking Nogo-B, systemically or specifically in the endothelium, are resistant to the onset of pathological hypertrophy. Furthermore, pharmacological inhibition of SPT with myriocin restores permeability, inflammation, and heart dysfunction in Nogo-A/B-deficient mice to wild-type levels; whereas SEW2871, an S1P
1
receptor agonist, prevents myocardial inflammation and dysfunction in WT banded mice. Our study identifies a critical role of endothelial sphingolipid biosynthesis and its regulation by Nogo-B in the development of pathological cardiac hypertrophy, and proposes a potential new therapeutic target for the attenuation or reversal of this clinical condition.
Abstract 89: 17beta-Estradiol-activated Estrogen Receptors Attenuate Cardiac Fibrosis Through Inhibition of Collagen I and III Expression in Female Hearts
Authors: Dworatzek Elke , Mahmoodzadeh Shokoufeh , Westphal Christina , Fliegner Daniela , Regitz-Zagrosek Vera
Published: June 2021

Objectives:
Female pressure-overloaded hearts show less fibrosis compared with males. 17β-Estradiol (E2) attenuates cardiac fibrosis in female mice. Whether this is mediated by direct E2-effects on collagen synthesis is still unknown. Therefore, we investigated the role of E2 and estrogen receptors (ER) on collagen I and III expression and analyzed involved mechanisms.

Methods:
Female C57BL/6J mice (7 weeks) underwent sham operation, ovariectomy (OVX), OVX with E2-supplementation (390mg E2-containing pellets) or placebo. After 2 weeks, animals underwent transverse aortic constriction (TAC) or sham surgery. Mice were sacrificed after 9 weeks. Collagen amount, collagen I and III protein in left ventricular tissue were detected by Sirius Red and antibody staining, respectively. Gene and protein expression were determined by quantitative Real-Time PCR and Western blot. Adult female rat cardiac fibroblasts were treated with E2 (10
-8
M), vehicle, ERα- and β-agonists (10
-7
M) for 24h or pre-treated with PD98059 for 1h. ER binding to the collagen I and III promoter was analyzed by chromatin immunoprecipitation assays.

Findings:
In female OVX mice, undergoing TAC surgery, E2-supplementation significantly reduced collagen deposition, collagen I and III mRNA and protein levels in comparison with mice without E2. In female rat cardiac fibroblasts, E2 significantly down-regulated collagen I and III mRNA and protein level. Specific ER-agonist-treatment showed that E2-mediated regulation of collagen I and III expression was mediated via activation of ERα, but not ERβ. Further, upon E2-treatment, ERα was phosphorylated at Ser118, which occurred by E2-induced activation of ERK1/2 signaling. Furthermore, we could show that ERα and ERβ bind to two putative half-palindromic estrogen response elements within the collagen I and III promoter in female cardiac fibroblasts.

Conclusion:
E2 inhibits cardiac collagen I and III mRNA and protein in female mice under pressure overload. Data from rat female cardiac fibroblasts suggest that this is mediated via E2-activated ERK1/2 signaling and ERα, which binds with ERβ to the collagen I and III promoter. Understanding of how E2/ER attenuate collagen I and III expression in pathological hypertrophy may improve therapy.
Abstract 99: IL10-inhibits Fibroblast Progenitor Cell-mediated Cardiac Fibrosis in Pressure-overloaded Myocardium
Authors: Verma Suresh K , Garikipati Venkata N , Krishnamurthy Prasanna , Benedict Cindy , Nickoloff Emily , Kishore Raj
Published: June 2021

Background:
Activated fibroblasts (myoFBs) play critical role in cardiac fibrosis, however, their origin in diseased heart remains uncertain. Recent studies suggest the contribution of bone marrow fibroblasts progenitor cells (BM-FPC) in pressure overload (PO)-induced cardiac fibrosis. Previously we have shown that interleukin-10 suppress PO-induced cardiac fibrosis, however, its role on inhibition of BM-FPC-mediated fibrosis is not known. Thus, we hypothesized that IL-10 inhibits PO-induced homing and transition of BM-FPC to myoFBs and therefore, attenuates cardiac fibrosis.

Methods and Results:
Cardiac fibrosis was induced in Wild-type (WT) and IL-10-knockout (KO) mice by transverse aortic constriction (TAC). TAC-induced left ventricular (LV) dysfunction and fibrosis were further exaggerated in KO mice. Systemic recombinant IL-10 administration markedly improved LV function and inhibited PO-induced cardiac fibrosis. PO-enhanced FPC (Prominin1
+
cells) mobilization and homing in IL-10 KO mice compared to WT mice. Furthermore, bone marrow transplantation (BMT) experiment was performed wherein WT marrow from GFP mice was repopulated in IL-10 KO mice. FPC mobilization was significantly reduced in BMT-IL10 KO mice compared to IL-10 KO mice after TAC. Furthermore, immunofluorescence result in BMT mice showed that subsets of myoFBs are derived from BM after TAC. To identify the molecular mechanism, wild type BM-FPC were treated with TGFβ
2
with or without IL10. IL10 treatment significantly inhibits TGFβ
2
-induced FPC to myoFBs transition. As miRNAs are key players in cardiac fibrosis, next we performed fibrosis-associated miRNA profiling using miRNA array kit. TGFβ
2
-induced miR-208, 155, 21 and 145 expression was markedly inhibited by IL-10.

Conclusion:
Taken together, our findings suggest that both reduced homing to heart and transition of FPC to myofibroblasts mediate anti-fibrotic effect of IL10 during PO-induced heart failure. Ongoing investigations using molecular approaches will provide a better understanding on the mechanistic and therapeutic aspects of IL10 on PO-induced cardiac fibrosis and heart failure.
Abstract 10: Neuroendocrine Dysfunction and Functional Disability Following Stroke
Authors: Kreber Lisa A , Singh Charan K
Published: June 2021

Comprehensive studies of the incidence of neuroendocrine dysfunction following stroke are lacking. The objectives of this study were to determine the incidence of neuroendocrine dysfunction in patients with stroke and to determine if growth hormone deficiency (GHD) contributes to disability following stroke.

Hormones were assessed in 130 patients with stroke (ischemic: n=65; hemorrhagic: n=65; M time since stroke=155 days; men=81; M age = 52). TSH, T3, T4, follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, testosterone, cortisol, and insulin-like growth factor-1 were assessed. All patients underwent glucagon stimulation testing (GST) to assess growth hormone (GH). Patients were then classified as having severe GHD, moderate GHD or normal GH based on the results of the GST. Functional disability was assessed with the Disability Rating Scale, Independent Living Scale (ILS), and the Mayo-Portland Adaptability Inventory-IV.

The majority of stroke patients had GHD (30% severe; 29% moderate). Patients with ischemic stroke had a higher prevalence of GHD (34% severe; 33% moderate) compared to patients with hemorrhagic stroke (26% severe; 23% moderate). The severe GHD group had significantly lower levels of FSH than the moderate GHD and normal GH groups (p<0.01). The moderate GHD group had significantly lower levels of LH than the severe GHD and normal GH groups (p<0.05) and significantly lower levels of T3 than the severe GHD and normal GH groups (p<0.05). The severe GHD group performed worse on the Activities of Daily Living and Behavior subscales of the ILS than the moderate GHD and normal GH groups (p<0.05).

GHD was more prevalent in patients with ischemic stroke, indicating that type of stroke may influence neuroendocrine functioning. Additionally, FSH, LH and T3 were lower in patients with GHD than in patients with normal levels of GH. The implications of these findings suggest that routine, comprehensive neuroendocrine testing may be warranted in individuals who have sustained a stroke. Patients with GHD were more functionally disabled in their ability to perform activities of daily living and to engage in socially appropriate behavior. These findings suggest that hormone deficiencies can influence disability post-stroke.
Abstract 109: Non-invasive Detection of Capillary Arteriovenous Shunting in Patients With Sickle Cell Anemia
Authors: Juttukonda Meher R , Donahue Manus J , Gindville Melissa C , Pruthi Sumit , Kassim Adetola A , Watchmaker Jennifer M , Hendrikse Jeroen , Jordan Lori C
Published: June 2021

Introduction:
High blood velocity can cause rapid erythrocyte transit through capillaries, reducing efficiency of oxygen delivery to tissue (capillary arteriovenous shunting). Arterial spin labeling (ASL) is an MRI technique that utilizes magnetic labeling of arterial blood water for CBF quantitation. Labeled water that traverses capillaries without exchanging with tissue leads to hyperintense venous signal indicative of arteriovenous shunting. We hypothesized that venous hyperintensity is present in sickle cell anemia (SCA) adults, correlates with flow velocity, and corresponds with clinical impairment and oxygen extraction fraction (OEF).

Methods:
ASL for shunting determination, TRUST for OEF measurement, phase contrast angiography for velocity assessment, and FLAIR / MRA for infarct / vasculopathy evaluation were performed at 3T in adults with SCA (n=36) and age- and race-matched controls (n=11). Three reviewers assessed for hyperintensity in the superior sagittal sinus on ASL images (Fig) and assigned scores of 0 (none), 1 (mild, focal), 2 (significant, focal), or 3 (significant, diffuse). Shunting scores were compared with the presence of clinical impairment (prior infarcts, stenosis>50%, or severe disease requiring transfusions) and OEF.

Results:
Interobserver agreement was excellent (Fleiss’ κ=0.91). Consensus shunting score in SCA adults (1.2±1.1) was higher (p<0.01) than controls (0.1±0.3), Median age 27.6 y, 57% F. Elevated shunting scores were observed in SCA adults with (1.23±1.07) vs. without (1.07±1.16) clinical impairment. A trend (p=0.068) for elevated OEF was observed in those with shunting scores ≥2 (0.44±0.07) vs. those with shunting scores ≤1 (0.40±0.07). Cervical flow velocity was elevated in subjects with shunting scores ≥2 (30.2±4.8 cm/s) vs. ≤1 (25.3±4.8 cm/s).

Conclusion:
Venous hyperintensity in ASL images may indicate capillary arteriovenous shunting and may reflect higher clinical impairment and elevated OEF.
Abstract 110: Caveolin-1 and Arterial Remodeling in Adult Moyamoya Disease
Authors: Chung Jong-Won , Choi Hyun Ah , Lee Mi Ji , Lee Joon Hwa , Park Moo-Seok , Yeon Je Young , Seo Woo-Keun , Kim Keon Ha , Kim Gyeong-Moon , Jeon Pyoung , Kim Jong-Soo , Hong Seung Chyul , Chung Chin-Sang , Lee Kwang Ho , Bang Oh Young
Published: June 2021

Background and purpose:
Moyamoya disease (MMD) is a unique cerebrovascular occlusive disease that is characterized by progressive stenosis and negative remodeling of the distal internal carotid artery (ICA). We hypothesized that caveolin-1, a protein that controls the regulation of endothelial vesicular trafficking and signal transduction, is associated with negative remodeling in MMD.

Methods:
We prospectively recruited 77 consecutive patients with MMD (49 bilateral and 28 unilateral MMD) diagnosed by conventional angiography. Seventeen patients with intracranial atherosclerotic stroke and no
RNF213
mutation served as controls. Distal ICA outer diameters were examined with high-resolution MRI. We evaluated whether the degree of negative remodeling in MMD patients was associated with
RNF213
polymorphism, caveolin-1 levels, or various clinical and vascular risk factors.

Results:
The
RNF213
variant was observed in 49 (63.6%) patients with MMD. The serum caveolin-1 (ng/mL) level was lower in MMD patients than in controls (0.47±0.29 vs. 0.86±0.68,
P
=0.034). The mean ICA diameter was 2.48±0.98 mm (range 0.00-4.76) for the 126 affected distal ICAs in MMD patients and 3.84±0.42 mm for asymptomatic ICAs in controls. After adjusting for possible confounders, male sex (coefficient, 0.396;
P
=0.029), clinical presentation with ischemic stroke (coefficient, -0.733;
P
<0.001), and caveolin-1 level (coefficient, 1.018;
P
<0.001) were independently associated with distal ICA diameter in MMD patients.

Conclusion:
Our findings suggest that caveolin-1 may play a major role in arterial negative remodeling in MMD patients. Future studies exploring caveolin-1 as a therapeutic target in MMD are warranted.
Abstract 115: Early Vessel Imaging At Primary Stroke Centers Improves Clinical Outcomes Of Patients Transferred For Thrombectomy.
Authors: McTaggart Ryan A , Yaghi Shadi , Baird Grayson , Cutting Shawna , Hemendinger Morgan , Siket Matthew , Haas Richard , Jayaraman Mahesh
Published: June 2021

Introduction:
We created an optimized ELVO transfer protocol for PSCs focused on three key steps: early CSC notification based on clinical severity threshold, obtaining vessel imaging at the PSC, and cloud-based image sharing without the use of telemedicine.

Methods:
Our regional PSC spokes were educated to execute the following protocol for patients presenting with a Los Angeles motor score ≥4: 1) notify the CSC on arrival 2) obtain CTA at time of initial NCCT and within 30 minutes of PSC arrival, and 3) implement a secure cloud-based remote viewing platform (LifeIMAGE). We reviewed all ICA and/or M1 occlusions transferred over an 11 month period, and divided the patients into two groups: those for whom the protocol was executed and those for whom it was not. Time from PSC arrival to start of angiogram was modeled using Kaplan-Meier estimation, with Wilcoxon test given no observations were censored. Favorable outcome (mRs 0-2 at 90 days) was modeled by protocol using a generalized linear model with a binary distribution.

Results:
Between 7/1/15 to 5/31/16 there were 70 patients transferred for thrombectomy. When executed, the PSC ELVO protocol reduced the median time for PSC arrival to CSC groin puncture from 151 [141.0, 166.0] to 111 [88.0, 130.0] minutes, a significant reduction of 40 minutes, p<.0001. This was primarily related to improvement in PSC door in door out time which reduced from a median of 104 [82.0, 112.0] to 64 [51.0, 71.0] minutes, a reduction of 40 minutes, p<.0001. When the protocol was followed, patients were twice as likely to have a favorable outcome (50% vs. 25%), p<.05. No differences were observed for age (p=.52), travel distance (p=.94), NIHSS (p=.49), sex (p=.44), and IV tPA use (p=.93) between the two groups.

Conclusion:
A standardized process at PSCs for suspected ELVO patients consisting of early CSC notification, CTA on arrival to the PSC and image sharing is easily implemented and significantly reduces the time to CSC groin puncture and improves outcomes.
Abstract 118: Paramedic-Administered Los Angeles Motor Scale identifies Ischemic Stroke with Large Vessel Occlusion and Intracranial Hemorrhage for Routing to Comprehensive Stroke Centers and Compares Favorably to Other Screening Methods
Authors: Noorian Ali Reza , Sanossian Nerses , Shkirkova Kristina , Liebeskind David S , Eckstein Marc , Stratton Samuel , Pratt Franklin D , Conwit Robin , Chatfield Fiona , Sharma Latisha K , Starkman Sidney , Saver Jeffrey L
Published: June 2021

Background:
Considering the recent advances in endovascular thrombectomy and advances in neurocritical care of patients with intracranial hemorrhage (ICH), there is an urgent need to develop tools for paramedics to identify patients likely to benefit from direct routing to comprehensive stroke centers (CSC). We report prospective validation of the Los Angeles Motor Scale (LAMS) performed by paramedics in the field, and compare its performance with other proposed prehospital LVO-identification scales.

Methods:
We analyzed all subjects enrolled in the NIH Field Administration of Stroke Therapy -Magnesium (FAST-MAG) trial transported directly to an academic center with a policy of performing immediate CTA or MRA imaging for all likely strokes. Prehospital LAMS was performed by paramedics prior to field enrollment. Hospital arrival (HA) LAMS and NIHSS were performed by trained study nurses after ED arrival. RACE, PASS, and 3i SS scales were calculated from NIHSS items. LVO in a proximal cerebral artery (ICA, MCA M1 & M2, Vertebral, Basilar and PCA P1 & 2) was determined by 3 vascular neurologists with expertise in neuroimaging. An LVO or ICH were considered as CSC appropriate patients.

Results:
Among 94 patients, age was 68 (±13) and 49% were female. Final diagnoses were acute cerebral ischemia in 71 (76%), intracranial hemorrhage in 18 (19%), mimic in 5 (5%). Overall, 48 patients (68%) had LVO, including MCA (30), ICA (14), basilar (1), vertebral (1) and PCA (2), and 66 (70%) were CSC-appropriate (LVO or ICH). In prediction of LVO, prehospital LAMS had the highest sensitivity (71%) and moderate specificity (54%). 3i-SS had the highest specificity (83%) but lowest sensitivity (40%). In prediction of CSC-appropriate patients, prehospital LAMS had the highest sensitivity (69%) and 3i-SS had the highest specificity (93%) but lowest sensitivity (37%). When comparing receiver operating curves, PM LAMS had AUC of 0.761, HA RACE 0.752, HA 3i SS 0.732, and HA PASS 0.712.

Conclusions:
Prehospital LAMS score of 4 or higher identified CSC-appropriate patients with good sensitivity and moderate specificity, and performed similar to or better than other proposed scales. LAMS is easy to administer and reproducible, and widely used currently by paramedics nationwide.
Abstract 12: Effect of Corticospinal Tract Injury on Motor Cortex Function and Connectivity After Stroke
Authors: Cramer Steven C , Cassidy Jessica M , Ingemanson Morgan , Srinivasan Ramesh
Published: June 2021

Background and Purpose:
Behavioral outcome after stroke is the product of both neural injury and neural function. Little is known about how injury to the corticospinal tract (CST) affects the function of motor cortex. The purpose of this study was to understand how subcortical injury to the CST affects function and connectivity of motor cortex.

Methods:
Patients with chronic hemiparetic stroke completed (1) a 3-minute resting-state EEG recording using a dense-array (256-lead) system, (2) a structural MRI scan, and (3) behavioral testing. Motor cortex
activity
was defined as EEG power within the high beta (20-30 Hz) frequency band commonly associated with motor network activity. Motor cortex
connectivity
was defined as coherence in the same frequency band. CST injury was defined as % lesion overlap with CST.

Results:
Of the 39 subjects (56 ± 14 years, 10 females, 15 ± 25 months post-stroke), none had injury to ipsilesional primary motor cortex (M1). Spearman correlation analyses revealed that increased CST injury was significantly related to reduced cortical activity in EEG leads overlying M1 (r= -0.48, p <0.002), dorsal premotor cortex (r= -0.41, p= 0.01), and supplementary motor area (r= -0.41, p= 0.01), but not in any other brain region, bilaterally. However, increased CST injury was not associated with any changes in M1 connectivity. Arm motor status (Fugl-Meyer score) tended to be associated with increased CST injury (r= -0.28, p= 0.08) but had no relationship with M1 connectivity.

Conclusions:
Increased CST injury after stroke is associated with decreased activity in those motor areas that issue descending fibers via this tract, a finding consistent with prior reports indicating that axonal injury modulates upstream function of surviving cortical elements. Increased CST injury was not associated with changes in M1 connectivity, suggesting a retained capacity for plasticity in support of behavioral recovery.
Abstract 125: Increased Endothelial Endoglin Gene Deletion and Co-deletion of EphrinB2 Enhance Brain Arteriovenous Malformation in Mice
Authors: Zhang Rui , Zhu Wan , Ma Li , Luo Man , Zhan Lei , Su Hua
Published: June 2021

Introduction:
Endoglin (Eng)
is an arteriovenous malformation (AVM) causative gene. In adult mice, global
Eng
deletion induced by a wild-type cre (cre) results in AVM formation in the brain angiogenic region. We hypothesize that using a codon-improved cre (icre) to increase
Eng
gene deletion in endothelial cells and co-deletion of the
EphrinB2
gene, a determinant of arterial endothelial differentiation, will enhance brain AVM severity.

Methods:
Eng
was globally deleted in adult
Eng
-floxed mice (
Eng
2f/2f
) using a rosa promoter driving estrogen inducible cre (Rosa-creER), or in endothelial cells using a platelet-derived growth factor b promoter driving estrogen inducible icre (
pdgfb-
icreER).
Pdgfb-
icreER was also used to mediate endothelial deletion of
EphrinB2
. An adeno-associated viral vector expressing vascular endothelial growth factor was injected into the brain to induce brain angiogenesis.

Results:
Compared with Rosa-CreER-mediated global
Eng
deletion,
pdgfb-
icreER-induced endothelial
Eng
deletion did not increase the number of abnormal vessels (P=0.39), but reduced vascular smooth muscle coverage (P=0.03) and increased hemorrhage (P=0.04) in the brain AVM lesion. Additional endothelial deletion of
EphrinB2
gene increased the number of abnormal vessels in the brain (P=0.08).

Conclusion:
These data indicate that a positive correlation exists between the degree of gene mutation in the endothelial cells and brain AVM severity, and that dysregulation of endothelial arteriovenous specification enhances AVM formation and progression.