bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 75: Ischemic Heart Failure is Exacerbated in CD39-null Mice
Authors: Novitskaya Tatiana , Wheeler Debra G , Xu Zhaobin , Chepurko Elena , Zhang Bo , Koch Walter , Dwyer Karen M , Cowan Peter J , Robson Simon C , Gao Erhe , Gumina Richard J
Published: July 2021

Background:
CD39 (ectonucleoside triphosphate diphosphohydrolase) is a nucleotidase expressed on endothelial cells, vascular smooth muscles cells, and leukocytes. CD39 plays a key role in vascular homeostasis, hydrolyzing extracellular ATP and ADP. CD39 has been shown to be important in models of ischemic preconditioning and cardiac ischemia reperfusion. However, the effect of CD39 activity on functional recovery of heart after myocardial infarction (MI) has not been evaluated.

Hypothesis:
Genetic ablation of CD39 expression exacerbates post-myocardial infarction cardiac function and fibrosis.

Methods:
Wild-type (WT) and CD39-null mice were subjected to coronary artery ligation. Cardiac function and protein evaluation of fibrotic markers was performed at day 28 post-MI.

Results:
Evaluation at Day 28 post-MI revealed that while mice of both genotypes had similarly reduced ejection fraction and equally compromised contractile function (dP/dtmax), there was a more pronounced negative effect on lusitropy (dP/dtmin) and increased left ventricular end-diastolic pressure in CD39-null mice. Therefore, cd39 gene ablation associates with the development of worsening cardiac performance. Histological analysis revealed increased collagen deposition and abundance of alpha-smooth muscle actin (αSMA) positive interstitial cells in the CD39-null hearts compared to WT hearts. To quantify these findings immunoblot analysis for collagen and αSMA was performed. We found that collagen and αSMA were increased at Day 28 post-MI, in CD39-null hearts compared to WT hearts.

Conclusion:
CD39 ablation has detrimental effects on post-MI recovery, resulting in diminished cardiac performance and increased fibrosis.
Abstract 123: Increased Susceptibility of Mitochondria to Permeability Transition Pore Opening in Alcoholic Cardiomyopathy
Authors: Emelyanova Larisa , Holmuhamedov Ekhson , Ryan Sean , Kraft Kelsey , Edwards Stacie , Negmadjanov Ulugbek , Holmuhamedov Alisher , Rizvi Farhan , Ross Gracious R. , Shi Yang , Tajik A. Jamil , Jahangir Arshad
Published: July 2021

Introduction:
Although generations of reactive oxygen species and mitochondrial dysfunction have been implicated in pathogenesis of alcoholic cardiomyopathy (ACM), molecular target(s) responsible for myocardial dysfunction in ACM are not well known.

Objective:
To determine the impact of chronic alcohol exposure on mitochondrial oxidative phosphorylation system (OXPHOS), permeability transition pore (mPTP) opening, and oxidative stress using a rat model of ACM.

Methods:
Sprague Dawley male rats (1 mo old) were exposed to alcohol (7.5% ethanol) for 3 months to develop ACM. Activity of OXPHOS was assessed enzymatically and by measuring mitochondrial oxygen consumption rate (OCR). The mPTP opening was determined by monitoring abrupt release of Ca
2+
after exposure of mitochondria to Ca
2+
. Western blot and RT-PCR were used to assess the expression of OXPHOS and mPTP protein components and corresponding genes. Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were determined in heart tissue homogenates.

Results:
There was no significant change in OCR or activity of OXPHOS complexes I-V, despite a decrease in complex V protein and
Atp6v1e2
gene expression (-1.7-fold, p<0.01) in Alc rats. Mitochondria from Alc rats were more sensitive to Ca
2+
-induced mPTP opening. This was associated with increase in protein expression level of adenine nucleotide translocase 1/2 and voltage dependent anion channel 1. There was no difference in MDA or 4-HNE levels.

Conclusion:
Increased sensitivity of mitochondria to mPTP opening during long-term alcohol consumption may compromise cardiac energetic reserves and contractility, leading to ACM development and progression.
Abstract 130: Developing Optogenetic Tools for Mitochondria and sarco/endoplasmic Reticulum Interactions
Authors: Yazawa Masayuki , Shi Fan
Published: July 2021

Interaction between mitochondria and sarco/endoplasmic reticulum (SR/ER) is essential for cardiac calcium handling, metabolism and autophagy. Here we present novel light-induced systems to control the interaction between mitochondria and SR/ER in mammalian cardiomyocytes. To develop this system, first we have tested and optimized multiple light-induced hetero dimer domains in mammalian cell lines, HEK 293T and NIH 3T3 cells. Using electron and confocal microscopes with an optimized system, next we confirm that blue light (~450-470nm, shot illumination ~1sec) could induce coupling of SR/ER with mitochondria. In addition, we developed reversible and non-reversible types of light-induced coupling of mitochondria and SR/ER for further applications in human and mouse cardiomyocytes with viral transduction. The new optogenetic tools will help us study the molecular mechanisms underlying cardiac calcium signaling, metabolism and autophagy.
Abstract 174: Regulation of Kv11.1 C-terminal Isoform Expression by Polypyrimidine Tract Binding Protein
Authors: Gong Qiuming , Goldys Anastasiya , Zhou Zhengfeng
Published: July 2021

The KCNH2 gene encodes the Kv11.1 potassium channel that conducts the rapidly activating delayed rectifier current in the heart. KCNH2 pre-mRNA undergoes alternative processing. Intron 9 splicing leads to the formation of a functional, full-length Kv11.1a isoform, and polyadenylation within intron 9 generates a non-functional, C-terminally truncated Kv11.1a-USO isoform. In this study we investigated the developmental regulation of Kv11.1 isoform expression. We showed that Kv11.1a expression was lower than that of Kv11.1a-USO in the adult heart, but the levels of Kv11.1a and Kv11.1a-USO were similar in the fetal heart. We studied the effect of polypyrimidine tract binding protein (PTB) on the alternative processing of KCNH2 pre-mRNA. PTB is an RNA-binding protein well known for its role in the regulation of alternative splicing. Recently, PTB has been shown to regulate polyadenylation. We showed that PTB increased Kv11.1a isoform expression and decreased Kv11.1a-USO isoform expression by the RNase protection assay and immunoblot analysis. In patch-clamp experiments, we found that PTB significantly increased Kv11.1 current. Our findings suggest that the relative expression of Kv11.1 C-terminal isoforms can be regulated by PTB. It has been reported that PTB protein abundance is progressively reduced during postnatal heart development. Thus, PTB may play an important role in developmental regulation of Kv11.1 isoform expression in the heart.
Abstract 203: Glucose Stimulated Endothelial Microparticles Increase Endothelial Cell Apoptotic Susceptibility
Authors: Bammert Tyler , Hijmans Jamie , Reiakvam Whitney , Levy Ma’ayan , Stockelman Kelly , Bruster Lillian , Goldthwaite Zoë , Greiner Jared , DeSouza Christopher
Published: July 2021

Clinical interest in endothelial cell-derived microparticles (EMPs) has increased due to their role in the pathogenesis of vascular disease. Although released by the endothelium, EMPs have autocrine properties that can significantly impact endovascular health. Hyperglycemic conditions, such as diabetes, are known to stimulate EMP release; however, the effects of these glucose-related microparticles on endothelial cell function are not well understood. High glucose concentrations induce endothelial cell apoptosis through a caspase-3-dependent mechanism. The aim of this study was to determine the effect of EMPs derived from a hyperglycemic condition on endothelial cell susceptibility to apoptosis. Human umbilical vein endothelial cells (HUVECs) were cultured (3
rd
passage) and plated in 6-well plates at a density of 5.0 x 10
5
cell/condition. Cells were incubated with RPMI 1640 media containing 25mM D-glucose (concentration representing a diabetic glycemic state) or 5mM D-glucose (control, normoglycemic, condition) for 48 h to generate EMPs. EMPs derived from both conditions were pelleted by centrifugation and resuspended in culture media. EMP identification (CD144
+
expression) and number was determined by flow cytometry. HUVECs (2 x10
6
cells/condition) were treated with EMPs (2:1 ratio) generated from either the hyperglycemic or normoglycemic conditions for 24 h. Thereafter, cells were treated with staurosporine (1μmol/L) for 3 h at 37°C and biotin-ZVKD-fmk inhibitor for 1 h at 37°C. Intracellular concentration of active caspase-3 was determined by enzyme immune assay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. Hyperglycemic EMPs resulted in significant increase in basal (1.5
+
0.1 vs 1.0
+
0.1 ng/mL) and staurosporine-stimulated (2.2
+
0.2 vs 1.4
+
0.1 ng/mL) caspase-3 activity compared with normoglycemic EMPs. Additional, the expression of miR-Let7a was markedly reduced (~140%) in response to hyperglycemic EMPs (0.43
+
0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell apoptotic susceptibility. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let7a expression.
Abstract 209: Association Between Cannabis Use and TakoTsubo Cardiomyopathy (TTC): Analysis from the NIS 2012 - 2014
Authors: Alliu Samson , Adejumo Adeyinka , Adegbala Oluwole , Namana Vinod , Chetana Pendkar , Yevgeniya Borovinskaya , Hecht Melvyn , Wolf Lawrence , Kamholz Stephan , Hollander Gerald , Shani Jacob , Durojaiye Modupeolowa
Published: July 2021

Background and Objective:
Marijuana use causes catecholamine surge with consequent tachycardia and elevation of both systolic and diastolic blood pressure. It is unclear if the catecholamine surge associated is sufficient to cause left ventricular wall apical ballooning (TakoTsubo Cardiomyopathy (TTC)). Given the similarity in the pathophysiology of TTC and mechanism of action of cannabis, we sought to investigate if there is any association.

Methods:
We obtained data from the HCUP-NIS of all patients older than 45 years hospitalized between 2012 - 2014. Our main outcome was diagnosis of TTC, and main exposure variables was cannabis use both identified using the ICD-9 codes. Using the SURVEYLOGISTICS procedure, we performed logistic regressions to estimate the odds of TTC diagnosis and in-hospital mortality among cannabis users adjusting for demographics, comorbidities, and other recreational drugs.

Results:
Of the 7,805,400 hospitalized patients who were > 45 years, 10,160 (0.1%) had a diagnosis of TTC, 54,311 (0.7%) were nondependent cannabis user and 5,045 (0.1%) were dependent cannabis users. We observed a significant association between TTC and nondependent cannabis use (OR 1.35, 95% CI: 1.10-1.65), but the association was nonsignificant for dependent cannabis use. After adjusting for potential confounders such as age, race, gender, comorbidities, cocaine, amphetamine and alcohol, nondependent cannabis use was associated with a 2-fold increased odds of TTC (AOR 2.00, 95% CI: 1.61-2.40). However, the association remained nonsignificant for dependent cannabis users (AOR 0.70, 95% CI: 0.25-1.92). Also, among patients diagnosed with TTC, there was no significant difference in the odds of in-hospital mortality among cannabis users (dependent and nondependent) when compared to nonusers (AOR 1.04, 95% CI: 0.39 - 2.70).

Conclusion:
In our study population, nondependent cannabis use was associated with significantly increased odds of TTC. However, among patients with TTC, in-hospital mortality rate was the same irrespective of cannabis exposure.
Abstract 252: The Capsaicin Sensitive Afferent Neuron Innervating Skeletal Muscle is Abnormal in the Mdx Mouse
Authors: Li Qinglu , Garry Mary
Published: July 2021

Duchenne muscular dystrophy (DMD) is a severe type of muscular dystrophy caused by a mutation of the dystrophin gene at locus Xp21, located on the short arm of the X chromosome. Muscle wasting and weakness are common in DMD and in the murine mdx model. We previously demonstrated Group III and IV afferent neurons, which innervate skeletal muscle and control blood pressure and heart rate in response to exercise, are abnormal in settings of ischemia and atrophy; such as cardiomyopathy. We hypothesized that these afferent neurons would also display abnormalities in the mdx mouse. To test this hypothesis, we developed a decerebrate mouse model using 10 wk and 6 mo old male BL10 WT and MDX mice to test mean arterial pressure (MAP) responses to intra-arterial capsaicin (IA-Cap; a specific stimulant of group IV afferent neurons). Mice were anesthetized and MAP was continuously recorded with a pressure transducer in the left carotid artery after which the animal was rendered decerebrate. Following decerebration, anesthesia was discontinued and IA-Cap (0,003-1ug/100ul) was delivered via the left common iliac artery. In rats, we have demonstrated this to be a valid model for evaluating MAP responses to activation of metabolically active afferent neurons. We observed that MAP increased in a dose-related fashion in both 10wk and 6 mo old WT and MDX, while 10 wk old MDX mouse had a normal response, the 6 months MDX mouse response was significantly blunted when compared to WT. To test whether these abnormalities are related to the onset of cardiomyopathy, Echocardiography was performed using 6 months old BL10 WT and MDX mice, no abnormality was found in terms of LV dimensions and function in MDX mice comparing with WT mice. Further studies will be performed to determine whether these abnormalities are inherent to changes in the skeletal muscle of the mdx mouse. We conclude that this murine model displays pressor responses to IA-Cap, similar to the rat and that MDX mice have a blunted blood pressure response to IA-Cap. These results indicate that abnormalities exist within the skeletal muscle afferent neurons in the mdx model.
Abstract 257: Endogenous Activating Transcription Factor 6 Preserves Heart Structure and Function in a Mouse Model of Myocardial Infarction-induced Heart Failure
Authors: Stauffer Winston T , Azizi Khalid , Blackwood Erik A , Kaufman Randal J , Glembotski Christopher C
Published: July 2021

Rationale:
The ER stress response is activated by the accumulation of misfolded, toxic proteins in the endoplasmic reticulum (ER), and upregulates proteins that restore ER protein-folding capacity. The ER-transmembrane protein, activating transcription factor 6 (ATF6) senses ER stress and responds by transcriptionally inducing many of these genes and is thus a key component of the adaptive ER stress response. We previously showed that in the heart, ischemia activates ATF6. Furthermore, transgenic mouse hearts expressing a conditionally activated form of ATF6, and subjected to
ex vivo
ischemia/reperfusion, exhibited preserved heart function and smaller infarcts. Our lab also showed that by serving as a novel inducer of a global anti-oxidant gene program, endogenous ATF6 limits cardiac damage caused by reactive oxygen species during reperfusion. However, the effect of endogenous ATF6 in the failing heart is not known. Given that acute ischemia caused by occlusion of the coronary arteries is the cause of myocardial infarction (MI), we hypothesized that endogenous ATF6 limits infarct size and preserves heart function during MI. Additionally, since deleterious cardiac remodeling and heart failure can be long-term consequences of MI, we hypothesized that ATF6 can mitigate these effects.

Objective/Methods:
To examine the role of endogenous ATF6 in heart failure,
in vivo,
we used a mouse model of MI-induced heart failure in mice with a global deletion of the ATF6 gene (ATF6 KO). Infarct size was measured by TTC staining and heart function was observed via longitudinal echocardiogram.

Results:
We found that following infarction, ATF6 KO mouse hearts had larger infarcts compared to control. Thus, ischemic cardiac tissue in the peri-infarct region requires ATF6 to limit cardiac myocyte death. Interestingly, ejection fraction following MI decreased more over 13 weeks in ATF6 KO mice relative to control. While control and ATF6 KO mouse hearts hypertrophied to a similar degree, KO mice showed greater cardiac dilation.

Conclusions:
Together these findings show for the first time that endogenous ATF6 acts to preserve heart structure and function in an MI model of heart failure, suggesting that ATF6 may be a viable therapeutic target for treatment of this disease.
Abstract 258: Pulmonary Overexpression of SERCA2A Improves Electrical Dysfunction and Suppresses Arrhythmias in Monocrotaline-induced Right Ventricular Failure
Authors: Strauss Benjamin , Sassi Yassine , Turnbull Irene C , Bueno-Beti Carlos , Kohlbrenner Erik , Hajjar Roger J , Hadri Lahouaria , Akar Fadi G
Published: July 2021

Background:
Pulmonary Arterial Hypertension (PAH) is a major cause of mortality due, in large part to right ventricular (RV) failure. Its electrophysiological (EP) effects however are poorly defined. Recently, a gene therapy approach targeting SERCA2a (S2A) to the lungs improved RV mechanical function in a rodent PAH model. Whether this approach improves myocardial EP properties remains unknown.

Methods:
Male Sprague Dawley rats received a subcutaneous injection of monocrotaline (MCT 60mg/kg) leading to PAH induced RV failure. Three wks later, rats underwent intratracheal delivery of aerosolized AAV1.S2A (1E11 gc, N=6) or no treatment (MCT, N=7). Age matched rats served as controls (CTRL, N=5). The EP substrate and risk of VT were assessed using high resolution optical action potential (AP) mapping in ex vivo perfused hearts.

Results:
MCT (6/7) but not CTRL (0/5) hearts were prone to pacing-induced VT (P<0.01). S2A gene therapy markedly suppressed the incidence of VT to <15% (P<.05 vs MCT). Investigation of the EP substrate revealed complete reversal of slow myocardial conduction in S2A treated compared to untreated MCT rats
(Fig A, B)
. AP duration (APD) and heterogeneity were increased in MCT and partially reversed by AAV1.S2A. Underlying the rise in heterogeneity was selective APD prolongation on the RV side causing >90 degree clockwise shift in the orientation of the transepicardial gradient, an effect which was not reversed by AAV1.S2A
(Fig C arrows)
.

Conclusion:
S2A gene therapy to the lungs ameliorates PAH induced EP remodeling and arrhythmia propensity. Our findings highlight for the first time the utility of a non-cardiac gene therapy approach for arrhythmia suppression.
Abstract 276: The Role of Diacylglycerol Acetyltransferase 1 and 2 in Cardiac Metabolism and Function
Authors: Roe Nathan D , Handzlik Michal K , Li Tao , Tian Rong
Published: July 2021

Cardiac triglyceride (TG) plays an important role in myocardial metabolism. TG synthesis is catalyzed by diacylglycerol:acetyltransferase (DGAT). Enhancing cardiac TG synthesis and turnover, by means of overexpression DGAT1, has protected hearts against stresses while blocking the TG turnover causes cardiomyopathy. In the meantime DGAT inhibitors are being developed for lipid lowering therapy, raising concerns whether DGAT inhibition affects cardiac function. Here we determined the role of the two cardiac DGAT isoforms in TG synthesis and turnover in the heart and their contribution to cardiac fatty acid metabolism. Using an inducible cardiac specific DGAT1 deletion mouse (iKO) together with DGAT2-specific inhibitor, we were able to achieve graded inhibition of TG synthesis and turnover as determined by
13
C-NMR spectroscopy of isolated perfused mouse heart. The iKO heart has normal TG level (CON 5.7±1.2 vs. 6.9±0.7 μg/mg wwt) and perfusing hearts with glucose (5.5mM), fatty acids (0.4mM) and lactate (1.2mM) for 1hr did not change the TG content in control (CON, pre-perfusion 3.9 ± 0.6 vs. post-perfusion 4.5 ± 0.7 μg/mg wwt), iKO (3.8±0.6 vs. 4.2±0.5 μg/mg wwt) or iKO+DGAT2 inhibitor (3.8±0.6 vs. 5.0±0.8 μg/mg wwt). Relative to CON, the rate of
13
C labeled fatty acids incorporation into the TG pool decreased by 32% in iKO (AUC 5.170 of 7.547, p<0.05) which was accompanied by an increase the oxidation of exogenous fatty acids (relative FAO: 48.5±5.3 for CON vs. 67.0±4.1% for iKO, p<0.05). Cardiac function, assessed by echocardiography (FS: CON 47.5±1.3 vs. iKO 45.8±3.0%, p>0.05) or by rate pressure product of the isolated perfused heart (CON 39149±1047 vs. iKO 40836±3424 bpm*mmHg, p>0.05) is normal in iKO hearts and remained unchanged after treatment with the DGAT2 inhibitor (37083±8507 bpm*mmHg). Coinhibition of DGAT1 and 2 abrogated
13
C labeled fatty acids incorporation into the TG pool by 58% (AUC 5.042 of 11.82, p<0.05) and suppressed expression of PPARα target genes relative to non-treated control hearts (p<0.05). Taken together, our data show that both DGAT1 and 2 contribute TG synthesis in the heart. Inhibition of both isoforms abrogated TG synthesis and reduced PPARα activity in the heart but did not affect cardiac function in isolated perfused heart.
Abstract 284: Elevation of Cardiac NAD+ Levels Improves Mitochondrial Function in Mice Lacking Sirtuin 3
Authors: Walker Matthew A , Tian Rong
Published: July 2021

The mitochondria rely heavily on the ratio between NAD+ and its reduced form NADH to maintain proper function and generate 95% of cardiac cellular energy. Sirtuin 3 (SIRT3) is the major mitochondrial deacetylase and its activity level has been linked to the NAD+/NADH ratio. Numerous studies have shown that deletion of SIRT3 results in hyperacetylation and impairment of mitochondrial enzymes involved in fatty acid metabolism and in the mitochondrial respiratory chain. Because impairments in these processes have shown to be associated with decreases in the NAD+/NADH ratio and contribute to deficits in energy production, we hypothesized that the SIRT3 knockout mice (SIRT3
-/-
) would have a decreased NAD+/NADH ratio and that normalizing the ratio would improve mitochondrial respiratory function and inner membrane potential. In the present study, we observed increases in both NAD+ (1.1-fold, n=3, p<0.05) and NADH (1.5-fold) in cardiac tissue from 14-week old SIRT3
-/-
mice compared to wild-type (WT) controls, resulting in a 26±2.2% reduction in the NAD+/NADH ratio. These changes correlated with decreased Complex I ADP stimulated respiration (173±16 vs 118±14 nmolO
2
/min/mg for WT and SIRT3
-/-
, respectively, n=3, p<0.05) and suppressed mitochondrial membrane potential. Intraperitoneal injection (I.P.) of Nicotinamide Riboside (NR) increased NAD+ levels in cardiac tissue lysates (WT 1.5-fold and SIRT3
-/-
1.4-fold, n=5, p<0.05) and in mitochondria isolated (WT 1.9-fold and SIRT3
-/-
1.7-fold) from the mice with no significant changes in NADH levels. Therefore, the NR I.P. injections normalized the NAD+/NADH ratio, partially restored the Complex I supported mitochondrial respiration (123±11 vs 157±8 nmolO
2
/min/mg for vehicle and NR treated SIRT3
-/-
mice, respectively, n=3, p<0.05), and improved mitochondrial membrane potential in the SIRT3
-/-
mice. These results suggest that increasing cardiac NAD+ levels can rescue mitochondrial dysfunction independent of SIRT3 protein deacetylation and warrants further investigation. The next step will be to test whether increasing cardiac NAD+ levels can improve mitochondrial function and reduce injury in SIRT3
-/-
mice subjected to chronic heart stress induced by transverse aortic constriction surgery.
Abstract 299: Cardioprotection from Ischemia/Reperfusion Injury by β-Arrestin-Biased β2-Adrenergic Receptor Activation
Authors: Grisanti Laurel A , de Lucia Claudio , Gao Erhe , Koch Walter J , Benovic Jeffrey L , Tilley Douglas G
Published: July 2021

β-Adrenergic receptors (βAR) are important regulators of cardiac function in the normal and failing heart. Activation of the β1AR subtype increases contractility and cardiomyocyte death whereas the β2AR can promote survival. In response to cardiac injury, increased catecholamines activate and downregulate β1AR thus, β-adrenergic receptor antagonists or β-blockers are commonly prescribed in to restore β1AR expression and preserve contractility. However, promoting the pro-survival effects of β2AR, which are known to occur in part through β-arrestin (ARR)-dependent signaling, may be a beneficial therapeutic strategy. Pepducins have been developed based on the intracellular loop (ICL) domains of β2AR to activate either Gαs- or βARR-dependent β2AR signaling pathways. We hypothesized that pepducin-mediated engagement of βARR-dependent β2AR signaling in the heart would be therapeutically advantageous following ischemia/reperfusion (I/R) injury. To test this, wild-type (WT) C57BL/6 mice received three intracardiac injections of either a βARR-biased pepducin (ICL1-9) or a scrambled control pepducin at the time of ischemia (30 min) followed by reperfusion. Assessment of cardiomyocyte death 24h post-I/R using TUNEL staining showed a decrease in cell death in animals treated with ICL1-9 when compared to scrambled pepducin, correlating with decreased infarct size and improved cardiac function as measured by echocardiography. Assessment of cardiac function at later time points showed that the cardioprotective effects of ICL1-9 were preserved over time and resulted in decreased cardiac remodeling. Although βARR1KO mice displayed similar cell death, infarct size, and contractility changes following I/R as their WT counterparts, they did not manifest a cardioprotective response from ICL1-9 treatment, indicating that βARR signaling is essential in relaying ICL1-9-dependent cardioprotection. These results demonstrate that pharmacologically-mediated activation of βARR-biased β2AR signaling provides a therapeutic benefit in the context of I/R-induced injury and may provide an improved strategy for the treatment of acute cardiac injury.
Abstract 331: Genome-wide Analysis of Cardiac and Cardiac Fibroblasts Regulatory Elements Reveals Combinatorial Control of Gene Expression
Authors: Golan Lagziel Tal , Caspi Lilac , Lewis Yair , Kehat Izhak
Published: July 2021

The mammalian body contains several hundred cell types that share the same genome, but can express distinct gene signatures. This specification of gene expression is achieved through the activity of cis-regulatory genomic elements (CRE), such as enhancers, promoters, and silencers. The Assay for Transposase-Accessible Chromatin followed by sequencing (ATAC-seq) can identify nucleosome evicted open chromatin, an established marker of regulatory regions. Using a differential ATAC-seq approach, coupled with RNA-seq, H3K27ac ChiP-seq, and computational transcription factor (TFs) binding analysis we comprehensively mapped cell-type and condition specific cis regulatory elements for cardiac fibroblasts and cardiomyocytes, and outlined the TFs that control them. We show that in cardiomyocytes six main transcription factor groups, that control their own and each other’s expression, cooperatively bind discrete distal enhancers that are located at a variable distance from the transcription start site of their target genes. None of these factors is entirely tissue specific in expression, yet various combination of binding sites for these factors, densely clustered within a nucleosome length of genomic stretch make these CREs tissue specific. Multiple tissue specific CREs in turn, are clustered around highly tissue specific genes, and multiple factors, acting from the same and from different CREs can converge on these genes to control their tissue specific expression. Together our data puts forward a mechanistic multi-level combinatorial model for cardiac specific genes expression
Abstract 380: Mapping the i
n vitro
Interactome of the Cardiac Sodium-Calcium Exchanger 1
Authors: Carlson Cathrine R , Hafver Tandekile L , Wanichawan Pimthanya , Lunde Marianne , Martinsen Marita , de Souza Gustavo A , Sejersted Ole M
Published: July 2021

The sodium (Na
+
)-calcium (Ca
2+
) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the
SLC8A1
gene. In the heart, it maintains cytosolic Ca
2+
homeostasis, serving as the primary mechanism for Ca
2+
extrusion during relaxation. Dysregulation of NCX1 is observed in end-stage human heart failure. In this study we used affinity purification coupled with mass spectrometry in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: 1) anti-NCX1 against endogenous NCX1 and 2) anti-His

with His-TF-NCX1
cyt
recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature and 29 occurred in both screens. Selected protein partners were validated for binding to NCX1 expressed in HEK293 cells. A cardiac NCX1 protein-protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where cell communication and signal transduction formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in cardiovascular disease. Exploring the molecular mechanisms of these protein partners in future studies may aid in elucidation of NCX1 regulation and facilitate selective therapeutic targeting of NCX1.
Abstract 392: Impacto of Nifedipine on Polymorphonuclear Cells in Wounds
Authors: Brasileiro Augusto , Oliveira Dinaldo C , Oliveira Danielle , Amorim Jessica , Rocha Marina , Medeiros Bruno , Dantas Lais , Gadelha Maria Isabel , Nunes Vitor
Published: July 2021

Introduction:
Skin wounds are a problem and many occur due to vascular diseases. There is a need for drugs (low cost) that can heal wounds. We aimed to evaluate if the use of nifedipine in wounds is associated with recruitment of polymorphonuclear cells higher than placebo.

Hypothesis:
Nifedipine stimulates the recruitment of polymorphonuclear in wounds.

Methods:
We performed 32 wounds in pigs and locally applied placebo or nifedipine (1%, 10% or 20%). Wounds were evaluated macro and microscopically at 6 different moments. A logistic longitudinal model of mixed effects was applied. For this purpose, the response to moderate or marked polymorphonuclear cells was considered as a dependend variable, with the comparison groups (placebo, nifedipine 1%, 10% and 20%) as the explanatory variable. Based on the placebo group, the OR was estimated with its respective confidence interval for the nifedipine groups at the different doses. Value of p <0.05 was considered significant. This study was approved by the animal research ethics committee.

Results:
The table below shows the proportion of polymorphonuclear cells among the comparison groups over the 6 analyzed moments.

Conclusions:
Wounds treated with nifedipine had higher recruitment of polymorphonuclear, regardless of the concentration used. This drug has potential for recruitment of this type of cell and may have a beneficial effect on healing.
Abstract 404: NLR Family, Pyrin Domain-containing 3 Knockout Rescues Cardiac Dysfunction Induced by High-fat Diet Feeding
Authors: Peterson Matthew R , Haller Samantha , Ta Tracy , Bosch Luiza , Smith Aspen , Sanders Alyssa , Aquino Jelard , He Guanglong
Published: July 2021

NLR family, pyrin domain-containing 3 (NLRP3) is a pattern recognition receptor responsible for perpetuating an inflammatory response through production of pro-inflammatory cytokines IL-1β and IL-18. It has been implicated in the sustained inflammatory response in obesity and multiple cardiovascular disease conditions. In order to investigate NLRP3 as a potential therapeutic target in metabolic syndrome, C57BL/6 wild-type (WT) and NLRP3 knockout (NLRP3-\-) mice were fed a normal diet (ND; 12% fat chow) or a high fat diet (HFD; 45% fat chow) for 5 months. At 5 months, echocardiography and glucose tolerance tests (GTTs) were performed. Cardiac function assessed by fractional shortening (FS) was significantly impaired by HFD feeding in the WT group (0.335 HFD vs. 0.456 ND; p<0.05) but not in the NLRP3-\- (0.449 HFD vs. 0.492 ND; p>0.05). FS was higher in NLRP3-\-HFD than in WT-HFD (p<0.05). Two-dimensional analysis shows the FS difference between NLRP3-\-HFD and WT-HFD was primarily explained by the difference in left ventricular end-systolic dimension (0.2716 cm WT vs. 0.1883 cm NLRP3-\-; p<0.05). Glucose tolerance measured by area under the curve (AUC) was significantly impaired by HFD feeding for both WT (23183 ND vs. 57298 HFD; p<0.001) and NLRP3-\- (23197 ND vs. 44626 HFD; p<0.001), but significantly better in the NLRP3-\-HFD than in WT-HFD (p<0.01). HFD feeding increased fasting blood glucose (FBG) for both WT (97.7 mg
.
dl
-1
ND vs. 164.7 mg
.
dl
-1
HFD; p<0.01) and NLRP3-\- (80.50 mg
.
dl
-1
ND vs. 108.8 mg
.
dl
-1
HFD; p<0.05), but significantly less in NLRP3-\- mice (NLRP3-\- vs. WT; p<0.05). For GTTs, body weight was significantly higher in the WT than NLRP3-\- fed HFD (47.93 g vs. 36.5 g; p<0.001). Body weight explained 92% of variation in glucose tolerance (p<0.0001) and 69% of variation in fasting blood glucose (p<0.0001). WT-HFD averaged 1.31X heavier than NLRP3-\-HFD, while the AUC for the IGTT was 1.28X larger for the WT-HFD than NLRP3-\-HFD. Body weights were not significantly different between genotypes at the time of echo. The results suggest that knockout of NLRP3 may be protective against HFD induced cardiovascular dysfunction. A protective effect on glucose tolerance is not strongly supported.
Abstract 410: Rhein Administration Prevents Cardiac Fibrosis by Interfering with the TGF-β Pathway
Authors: Barbosa David , Wehmöller Melanie , Spinner Maximilian R , Rüther Ulrich , Ouwens Margriet
Published: July 2021

Fibrosis, which occurs in various heart diseases like acute myocardial ischemia and pressure overload, is triggered by the differentiation of fibroblasts into myofibroblasts. Dysregulation of this reparative mechanism results in excessive collagen accumulation leading to cardiac stiffness and impaired heart function. The aim of this study was to determine whether the rhubarb anthraquinone Rhein, a drug already used as treatment for chondroarthritis, prevents the transdifferentiation of cardiac fibroblasts. We observed that Rhein pre-treatment ameliorates the cardiac function and reduces adverse remodeling after acute myocardial infarction in mice,
in vivo
. In primary human cardiac fibroblasts, Rhein incubation dose-dependently inhibited the TGF-β-mediated upregulation of α-SMA, the master marker for myofibrolasts, and prevented the contraction of fibroblast-populated collagen gel lattices upon TGF-β stimulation. Further, Rhein reduced TGFβ-R1 expression in primary human cardiac fibroblast, resulting in decreased SMAD2 phosphorylation and blunting of the fibrogenic response. Furthermore, Rhein stabilized protein levels of SMAD7, a key inhibitor of TGF-β signaling. Collectively, these data show for the first time that Rhein administration prevents cardiac fibrosis
in vivo
and
in vitro
by blunting the TGF-β signaling pathway, and identify Rhein as potential therapeutic treatment to prevent excessive fibrosis and adverse remodeling in cardiac pathologies.
Abstract 435: Adrenergic Deficiency Disrupts Glucose Metabolism and ATP/NADH Production During Embryonic Heart Development
Authors: Peoples Jessica , Maxmillian Timmi , Le Quynh , Nadtochiy Sergiy M , Brookes Paul , Porter George , Ebert Steven
Published: July 2021

Cardiac energy demands increase during embryonic development, requiring activation of oxidative phosphorylation to convert ADP to ATP in mitochondria. We have recently shown that adrenergic hormones are required to maintain sufficient cardiac energy metabolism during embryonic development, but the specific mechanism(s) underlying this regulation are not known. Mouse embryos lacking the adrenergic hormones, norepinephrine (NE) and epinephrine (EPI), due to targeted loss of the
dopamine β-hydroxylase
(
Dbh
) gene, have markedly (>50-fold) decreased steady-state ATP/ADP ratios. Rates of ATP synthesis and hydrolysis did not differ between adrenergic-deficient and competent embryos suggesting the enzymatic machinery required for ATP production/consumption is functional. We hypothesized that adrenergic-deficient embryonic hearts are metabolically starved of nutrients leading to energy depletion. To identify changes in metabolism in adrenergic-deficient hearts, we performed LC-MS metabolomics, which showed decreases in all nucleotide triphosphates (NTPs) and NAD(H) confirming energy depletion. Additionally, products of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate dehydrogenase (G-6-PDH), and pyruvate dehydrogenase (PDH) were significantly diminished compared to controls, suggesting impaired activity. Enzymatic activities of GAPDH, G-6-PDH, PDH, and glycogen phosphorylase (GP); a well-known enzyme under adrenergic regulation, were measured from the rate of NAD(P)H production. GAPDH, G-6-PDH, and GP activities were significantly decreased (~80%, 40%, and 70% reduction, respectively) compared to controls. Interestingly, GAPDH, G-6-PDH, and GP protein levels, examined by western blot, did not differ from adrenergic-competent controls, thereby suggesting that adrenergic hormones regulate posttranslational activity of these enzymes. These results indicate that mitochondria are metabolically starved due to impairments in glycogenolysis, glycolysis, and pentose phosphate pathways. These findings reveal new mechanistic insights into global adrenergic regulation of major metabolic pathways during embryonic heart development.
Abstract 465: G3BP1 Supports Osteogenic Mitochondrial Antiviral Signaling - NFAT Transcriptional Relays in Aortic Vascular Smooth Muscle Cells
Authors: Gay Austin T , Ramachandran Bindu , Stabley John N , Cheng Su-Li , Behrmann Abraham , Li Li , Mead Megan , Williams Bart O , Chen Zhijian , Towler Dwight A
Published: July 2021

In vascular smooth muscle (VSM), LRP6, a Wnt co-receptor, inhibits arteriosclerotic noncanonical Wnt signals that activate protein arginine (Arg) methylation relays, converging on nuclear factor of activated T cells (NFAT). LRP6 deletion in VSM increases arteriosclerotic calcification and stiffness in low density lipoprotein receptor (LDLR)-deficient mice fed diabetogenic diets.

To study how Arg methylation mediates LRP6 actions, immunoaffinity mass spectrometry was performed on aortic VSM cell extracts from SM22-Cre;LRP6(fl/fl);LDLR-/- and LRP6(fl/fl);LDLR-/- mice. LRP6 deficiency altered Arg methylation of over 490 proteins, but only 22 exhibited increased monomethylation (MMA) with reduced dimethylation. One protein involved in atherosclerosis, G3BP1, exhibited a >30-fold increase in MMA of its C-terminal domain with LRP6 deficiency. Co-transfection confirmed that protein Arg methyltransferase 1 modified G3BP1 and was inhibited by LRP6. G3BP1 stimulated Frizzled-dependent NFAT transcription, again inhibited by LRP6. Nuclear NFATc4 and NFATc4 association with osteopontin (OPN) and alkaline phosphatase (TNAP) chromatin increased in LRP6-deficient VSM, and was reduced with G3BP1 deficiency. G3BP1 activation of NFAT required its nuclear transport domain and the MMA modified C-terminal domain to respond to Ddx58 - a modulator of mitochondrial antiviral signaling (MAVS) linked to aortic calcification. Ddx58 colocalized and synergized with G3BP1 in VSM to upregulate targets of noncanonical signaling, including OPN and TNAP. While VSM LRP6 deficiency increased Ddx58 levels, G3BP1 insufficiency reduces Ddx58 and osteogenic expression. RNAi targeting G3BP1, Ddx58 or NFATc4 reduced VSM osteogenic programs; VSM from MAVS-deficient mice exhibited reduced TNAP activity and osteogenic gene expression. Mass spectrometry data indicates that G3BP1 and Ddx58 form complexes with specific nucleoporins regulating nucleocytoplasmic transport.

Thus, G3BP1 is a target of MMA regulated by LRP6. G3BP1 promotes osteogenic signals in VSM, conveyed by a MAVS relay to regulate osteogenic transcription. Targeting G3BP1 and protein Arg methyltransferases may mitigate arteriosclerotic calcification in type 2 diabetes.
Abstract 469: Targeting Beta Adrenergic Receptor Resensitization Aids in Preservation of Receptor Function
Authors: Martellli Elizabeth E , Sun Yu , George John , Mohan Maradumane L , Starling Randall , Moravec Christine , Naga Prasad Sathyamangla V
Published: July 2021

Beta adrenergic receptor (βAR) down-regulation and desensitization are hallmarks of heart failure. Traditionally, it has been considered that increased desensitization mechanisms underlie βAR dysfunction in heart failure but it is not known whether resensitization of βARs is altered and is an integral contributor to heart failure. We have previously shown that resensitization is regulated by inhibition of PP2A by I2PP2A via PI3Kγ (Vasudevan et. al., 2011), the underlying mechanisms of I2PP2A binding to PP2A are not well understood. We used PyMOL software to find the binding interaction between PP2A and I2PP2A. Based on
in silico
predictions, we generated a mutant PP2A that when expressed would compete out I2PP2A and inhibit I2PP2A from binding to endogenous PP2A. Expression of PP2A mutant in β2AR expressing cells showed preservation of β2AR function following stimulation as measured by reduced β2AR phosphorylation, increased cAMP generation and increased phosphatase function. We also generated a small molecule from our
in silico
predictions that could target the interface of I2PP2A and PP2A binding to find that disruption of the PP2A/I2PP2A interaction underlies receptor function. We will use this small molecule to look at preservation of βAR function and amelioration of cardiac function. To test whether resensitization is altered in heart failure we used plasma membrane and endosomal fractions from non-failing and paired pre- and post-LVAD samples to show PI3K activity, PP2A activity, β2AR phosphorylation and adenylyl cyclase activity as a measure of recovery in βAR function. Our studies showed that endosomal fractions from human heart failure samples had elevated PI3K activity associated with reduced PP2A activity supporting the idea that βAR resensitization is inhibited in human heart failure samples. Since human heart failure samples have inhibited resensitization we tested the underlying mechanisms regulating βAR resensitization. Thus ongoing studies suggest that targeting the resensitization of βAR could provide beneficial cardiac remodeling in conditions of chronic mechanical overload and will be further discussed.
Abstract 473: Differential Regulation of Sk Channels by CamKII and Pyk2 Under Adrenergic Stimulation
Authors: Terentyeva Radmila , Polina Iuliia , Hamilton Shanna , Roder Karim , Koren Gideon , O-Uchi Jin , Terentyev Dmitry
Published: July 2021

Introduction:
Small conductance calcium (Ca)-activated K (SK) channels present in the plasmalemma of ventricular cardiomyocytes (VCMs) are dormant in health and become functional in cardiac disease. We recently showed that SK channels contribute to VCM repolarization in a rat model of hypertrophy induced by thoracic aortic banding (TAB), despite reduced expression levels, suggestive of increased activity of the channels. However, the mechanisms responsible for disease-associated functional upregulation of the channels are yet to be defined.

Hypothesis:
Functional recruitment of SK channels in cardiac hypertrophy is caused by enhancement of adrenergic signaling cascades.

Methods:
The effects of α1 adrenergic stimulation by phenylephrine (10 μM) + propranolol (100 μM) were studied in patch-clamped rat VCMs overexpressing rat SK2 after 48 hrs in culture. Calcium transients were recorded simultaneously using confocal microscopy and were used as a ramp [Ca] to assess possible changes in Ca sensitivity of the channel. Phosphorylation of SKs by Ser/Thr and tyrosine kinases, phosphorylation of Calmodulin at Thr-79, expression levels and phosphorylation of CaMKII and tyrosine kinase Pyk2 were assessed in cultured rat VCMs and freshly isolated VCMs from TABs and Shams using Western blot analysis.

Results:
WB analysis showed increased Ser/Thr and Tyr phosphorylation of SK2 in TABs. Alpha1 stimulation of rat VCMs overexpressing rat SK2 mimicked this pattern, increasing I
SK
at high voltages in parallel with the increase in Ca transient amplitude and reduction in Ca sensitivity of the channels. Overexpression of Pyk2 inhibitor CRNK did not restore I
SK
despite reduction in Ca transient amplitude because of attenuating α1-agonist mediated decrease in Ca sensitivity of SKs. Application of CaMKII inhibitor KN93 fully reversed α1-mediated I
SK
increase at high voltages without affecting Ca sensitivity of the channel.

Conclusion:
Upregulation of SKs in hypertrophic hearts is likely caused by CaMKII-dependent phosphorylation which reduces voltage-dependent block of the channels. Pyk2 inhibition may present therapeutic potential by attenuating negative effects on SK Ca sensitivity thereby increasing repolarization reserve diminished in cardiac disease.
Abstract 65: Anemia-related Heart Failure in Sub-saharan African Sickle Cell Disease Patients
Authors: Atanda Adebayo C , Aliyu Yahya , Atanda Oluwafunmilayo , Babadoko Aliyu , Suleiman Aisha , Mamman Aisha , Aliyu Zakari
Published: July 2021

Introduction:
Anemia has been implicated in heart failure. Existing literatures, involving predominantly African-Americans, suggests that Sickle Cell Disease (SCD) maybe linked to various cardiovascular complications including pulmonary hypertension and left venticular dysfunction. Peculiarly, our study involves exclusively Sub-Saharan population.

Method:
We conducted a cross sectional observational study of 208 hydroxyurea-naive consecutive SCD patients aged 10-52 years at steady state and 94 healthy non-matched controls who were studied in an out patient clinic in Sub-Saharan Africa. SCD patients were required to have electrophoretic or liquid chromatography documentation of major sickling phenotypes. Control group was required to have non-sickling phenotypes. Cardiac measurements were performed with TransThoracic Echo according to American Society of Echocardiography guidelines. Hemoglobin level was also obtained.

Results:
Hemoglobin level in SCD group (8.5+/- 1.5) was significant (P<0.001) compared to control (13.8+/- 1.7). Although SCD group had significantly higher values of left ventricular (LV) size, there was no qualitative evidence of LV dysfunction. SCD group had higher values of Ejection Fraction but not statistically significant. There was no evidence of LV wall stiffening to impair proper filling in SCD group, with the ratio of early to late ventricular filling velocities, E/A ratio elevated (1.7+/-0.4 compared to 1.6+/- 0.4; P=0.010). Right ventricular systolic pressure was determined using the formula of 4x Tricuspid Reugurgitant jet (TRV) square as an indirect measurement of Pulmonary arterial systolic pressure. SCD patients had significantly higher mean±SD values for tricuspid regurgitant jet velocity than did the controls (2.1±0.6 vs. 1.8±0.5; p= 0.001). Within the SCD group, there was no clear pattern of worsening diastolic function with increased TRV. Furthermore, E/A had a significant positive relationship with jet velocity in bivariate analysis (R=0.20; P=0.013).

Conclusions:
We were unable to demonstrate existence of anemia-associated left ventricular dysfunction in Sub-Saharan African with SCD. Further studies is required to highlight the reason behind this finding.