bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 317: Endoplasmic Reticulum Stress Alters the Localization of the Prolyl Hydroxylase Ogfod1 in Adult Mouse Cardiomyocytes
Authors: Harris Michael , Kennedy Leslie , Sun Junhui , Cockman Matthew , Ratcliffe Peter , Murphy Elizabeth
Published: October 2020

Prolyl hydroxylation is a post-translational modification that is known to regulate several key cell functions including translation and protein stability. Enzymes that catalyze prolyl hydroxylation belong to the 2-oxoglutarate- and iron-dependent oxygenase (2OGDO) family. A newly-identified member of the 2OGDO enzyme family, 2-oxoglutarate- and iron-dependent oxygenase domain-containing protein 1 (Ogfod1), catalyzes prolyl hydroxylation of ribosomal protein s23 (Rps23), a component of the 40S ribosome. To establish an
in vivo
role for Ogfod1, we ablated
Ogfod1
in mice and subjected isolated perfused hearts to ischemia and reperfusion and found
Ogfod1
ablation to be cardioprotective. The mechanisms by which these changes occur are currently unknown, so we investigated Ogfod1 regulation and mechanisms of cardioprotection. Previous work has shown that Ogfod1-mediated Rps23 prolyl hydroxylation occurs in the nucleus, where ribosomes are assembled. Additionally, Ogfod1 localizes to cytoplasmic stress granules in transformed cells exposed to endoplasmic reticulum (ER) stress. Based on these studies, we tested the hypothesis that Ogfod1 localization, and subsequently its function, change in response to stress. We induced ER stress by treating isolated adult cardiomyocytes with thapsigargin, and monitored Ogfod1 localization using immunofluorescence. Thapsigargin inhibits sarco/endoplasmic reticulum Ca
2+
ATPase (SERCA) which activates the unfolded protein response. In the absence of thapsigargin, Ogfod1 localized to the nucleus, peri-nuclear space, cytoplasm, and cell membrane. After stress induction, Ogfod1 nuclear- and perinuclear-localization decreased. This suggests that in murine adult cardiomyocytes subjected to ER stress, Ogfod1 is exported from the nucleus, potentially as a mechanism for down-regulating its activity. As Ogfod1 ablation has been found to be cardioprotective in mice, understanding Ogfod1 localization and regulation in the cardiomyocyte stress response may provide mechanistic insight that will be useful in developing treatments for stressors such as heart failure.
Abstract 318: Eicosapentaenoic Acid Improved Nitric Oxide Bioavailability and Reduced Nitroxidative Stress in Human Endothelial Cells in Contrast to Arachidonic Acid
In Vitro
Authors: Mason Preston , Dawoud Hazem , Sherratt Samuel , Libby Peter , Bhatt Deepak L , Malinski Tadeusz
Published: October 2020

Treatment with prescription, high dose, stable icosapent ethyl (IPE), which is eicosapentaenoic acid (EPA), an omega-3 fatty acid (O3FA), significantly reduced clinical events in high-risk patients with diabetes and other risk factors or cardiovascular disease (REDUCE-IT). Previous studies suggest that the benefits of EPA correlate positively with its levels and ratio to arachidonic acid (AA) in circulation. Unlike EPA, AA is an omega-6 fatty acid (O6FA) that, along with its metabolites, contributes to inflammation and diabetes. One mechanism of benefit of an increased EPA to AA ratio may be improved endothelial cell (EC) function, as evidenced by increased nitric oxide (NO) release and decreased nitroxidative (ONOO
–
) stress. In this study, human umbilical vein endothelial cells (HUVECs) were pretreated with EPA or AA at equimolar levels (10 μM) at various time points (4-24 hr) in 5% FBS. Following treatment, the cells were stimulated with calcium ionophore and assayed for the ratio of NO and ONOO
–
release, an indicator of eNOS coupling, using tandem porphyrinic nanosensors. ECs treated with EPA had significantly greater NO release following stimulation compared with vehicle at all time points, including 17% and 21% at 4 and 24 hr, respectively (p<0.05 and p<0.01) without changes in eNOS expression. By contrast, AA did not significantly improve NO production. ECs treated with EPA also showed a non-significant reduction in ONOO
-
release by 10% at 4 hr and 14% at 24 hr. EPA, but not AA, increased NO/ONOO
-
release ratio by 42% (4.03 ± 0.06 vs 2.83 ± 0.05;
p
<0.01) by 24 hr. Thus, EPA increased NO bioavailability in human ECs, unlike AA, due to improved eNOS coupling and reduced oxidative stress. These findings support a preferential benefit of EPA on endothelial function as compared to AA and supports further investigation.
Abstract 319: Tnf-α and Il-1β Decrease Myofibroblast Differentiation in 3d-cultured Mitral Valve Interstitial Cells
Authors: Zhu Amadeus , Grande-Allen Jane
Published: October 2020

Background:
Fibrosis contributes to many heart valve diseases such as calcific aortic valve disease, rheumatic heart disease, and secondary mitral regurgitation. Heart valve leaflets are populated by quiescent, fibroblast-like valve interstitial cells (VICs). During fibrosis, VICs differentiate into activated, myofibroblast-like cells that adversely remodel the extracellular matrix. Activated VICs overexpress α-smooth muscle actin (ACTA2/αSMA) and smooth muscle 22-α (TAGLN/SM22α) and display increased contractility. Tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1β) have been reported to either promote or inhibit fibrosis, depending on tissue type. Understanding how TNF-α and IL-1β affect VIC activation in the mitral valve of the heart could enable development of pharmaceutical treatments for heart valve diseases, which are currently managed surgically.

Methods:
To avoid artifactual activation on tissue culture plastic, VICs were encapsulated in biomimetic scaffolds consisting of polyethylene glycol (4% w/v) functionalized with protease-degradable (GGGPQGIWGQGK) and integrin-binding (RGDS) peptides. These 3D cultures were treated with 10 ng/ml TNF-α, 10 ng/ml IL-1β, or vehicle for 2 days in low-serum (1%) media. RNA and protein were measured via qRT-PCR, western blotting, and immunostaining. To measure contractility, VICs were encapsulated in collagen I (2.5 mg/ml) gels and allowed to contract freely for 2 days.

Results:
TNF-α and IL-1β significantly decreased RNA expression of ACTA2 (TNF-α: -91±6%, IL-1β: -99±1% change vs. vehicle) and TAGLN (TNF-α: -77±9%, IL-1β: -93±1% change). TNF-α and IL-1β also significantly decreased αSMA protein expression (TNF-α: -76±11%, IL-1β: -91±5% change) and the percentage of αSMA-positive cells (vehicle: 21±3%, TNF-α: 13±2%, IL-1β: 13±5% positive). Finally, TNF-α and IL-1β attenuated VIC-mediated collagen gel contraction (vehicle: 81±7%, TNF-α: 71±3%, IL-1β: 61±4% contraction).

Conclusions:
TNF-α and IL-1β decrease VIC activation in a 3D culture model of the mitral valve. These results reveal novel pathway targets for reducing fibrosis during mitral valve disease. Future work will use this model to study the downstream signaling events that drive VIC de-activation.
Abstract 323: Gamma-Enolase in Adult Cardiac Myocytes: A Study of Subcellular Localization and Post-Myocardial Infarction Modulation
Authors: Milcarek Andrew T , Dedkov Eduard I
Published: October 2020

Background:
Gamma-enolase, or neuron-specific enolase, is a multifunctional protein which is primarily found in neurons and neuroendocrine cells. Although the main role of gamma-enolase is associated with enzymatic activity during aerobic glycolysis, recently, it has been implicated in a variety of pathophysiological processes including inflammation, extracellular matrix degradation and cell survival after ischemic insult. Over the last decades, there have been a number of reports demonstrating the expression of gamma-enolase in the heart. Surprisingly, the latter finding has not received much attention in the past. Considering that this protein can play an important role in cellular adaptation to the ischemic environment we decided to examine the expression of gamma-enolase in the heart at different time points following a myocardial infarction (MI).

Methods:
A transmural MI was induced in male, middle-aged Sprague-Dawley rats by permanent left coronary artery ligation. The rats were euthanized 3 days, 1, 2, 4 and 8 weeks after MI and their hearts were processed into paraffin for immunostaining and quantitative morphometry.

Results:
We found that in post-MI heart gamma-enolase protein was expressed in cardiac myocytes of the remote myocardium as well as in the muscle cells surviving inside the transmural scar. On a subcellular level, the gamma-enolase expression overlapped with the I-bands of the sarcomeres. Despite the heterogeneity of the gamma-enolase fluorescence intensity among individual cardiac myocytes, there was no evident correlation between the size of the cells and the intensity of the gamma-enolase immunofluorescence in any region of the post-MI heart, although the immunofluorescence intensity of gamma-enolase in myocytes of the remote myocardium was always greater than that in myocytes surviving within the transmural scar. Most importantly, we determined that the intensity of gamma-enolase immunofluorescence recorded in cardiac myocytes has experienced an evident stepwise reduction at 1 and 8 weeks following MI.

Conclusions:
Taken together, our findings reveal that the cardiac myocytes of the adult rat heart express gamma-enolase and that the expression of this protein has been altered during post-MI cardiac remodeling.
Abstract 325: The Regulation of Endothelial Function Through Hmgcr/mevalonate Pathway Mediated Yap Activity
Authors: Liu Chun , Liu Yu , Chen Christopher , Ameen Mo , Yang Huaxiao , Shen Mengcheng , Rhee June Wha , Chen Ian Y , Sayed Nazish , Wu Joseph C
Published: October 2020

Background:
Cardiovascular diseases (CVD) are the leading cause of death in the United States. Statins, a class of hydroxy-methylglutaryl-coenzyme A reductase (HMGCR) inhibitors, have been proven to be effectively prevent and treat CVD by improving vascular functions independent of their cholesterol-lowering effect. However, the molecular mechanisms by which statins improve cardiovascular functions remain elusive. In this study, we used human induced pluripotent stem cells-derived endothelial cells (hiPSC-ECs) to explore the protective role of statins in the vascular system.

Methods and Results:
hiPSCs were generated from 3 healthy individuals and differentiated into endothelial cells using two different iPSC clones and two batches (4 biological replicates for each individual). hiPSC-ECs of each individual were treated with simvastatin or a vehicle control. The RNA-sequencing analysis was performed on 12 control and 12 statin-treated hiPSC-ECs. A total number of 2,580 differentially expressed genes (DEGs) were found in simvastatin-treated hiPSC-ECs. Gene enrichment analysis revealed that statin-upregulated DEGs were highly enriched in angiogenesis and anti-inflammation pathways. Interestingly, statin-downregulated DEGs were significantly enriched in epigenetic regulation and nucleosome assembly pathways, suggesting an epigenetic regulatory role of stains in vascular gene expression. To test this hypothesis, we further performed the Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq) analysis on vehicle or simvastatin treated hiPSC-ECs. Transcription factor (TF) binding motif analyses of ATAC-seq peaks using HOMER revealed that YAP/TEAD binding was the most significantly downregulated TF after statin treatment. Furthermore, we observed that statin can improve endothelial functions (e.g., angiogenesis and nitric oxide production) after the YAP activity was inhibited. The ChIP-seq analysis showed that statin downregulated the expression levels of genes associated with the endothelial-to-mesenchymal transition (EndoMT) by attenuating the binding capacity of YAP. As such, simvastatin effectively rescued diabetic vascular dysfunction mainly through inhibiting EndoMT.

Conclusion:
We found that statins can improve endothelial functions through attenuating the chromatin accessibility of EndoMT-related genes, a process tightly regulated by the activity of YAP. These findings will provide novel insights into the protective mechanisms of statins in the cardiovascular system beyond their cholesterol-lowering effects.
Abstract 326: Comparison of Patients’ Subjective Sense of Well Being and Their Cardiopulmonary Exercise Test Results
Authors: Bakhtiar Ayesha , Yousphi Adeel , Sheikh Adnaan , Yang Lan , Khan Shahkar A , Mahmood Esha , Mistry Hemanshi , Suleman Amer
Published: October 2020

Introduction:
The cardiopulmonary exercise test (CPET) helps assess exercise tolerance in patients. The CPET is a better predictor of general health status and functional capacity (fc) when compared with resting cardiac and pulmonary function testing measurements (1). The test makes use of a bicycle ergometer with the patient being fitted with ECG leads, blood pressure cuff, pulse oximeter, and a face mask which determines the amount of oxygen consumed and carbon dioxide released. The predicted peak oxygen consumption per unit body weight per unit time (VO2max) is calculated based on patient’s sex, age, weight and height (2). Fc is measured as a percentage of VO2max that was actually consumed.

Objective:
Our study evaluates any relationship between the CPET fc results and patient’s sense of overall well-being, which provides a subjective measure of their quality of life.

Methods:
In this retrospective study, we randomly selected 299 patients who visited the clinic using electronic medical records from May-June 2019. There were 260 females and 39 males, with ages between 16-82 years. Patient history was analyzed to look for “overall health rating” scores; how they felt about their overall health on a scale of 1 to 10, with 1 denoting feeling the worst and 10 indicating feeling the best of their health. Their CPET fc results were analyzed at the same time, where values <55% meant severely reduced fc, ≥55% but <70% meant moderately reduced fc, ≥70% but <80% meant mildly reduced fc, and ≥80% meant normal fc. Logistic regression was used to look for an association between the values of the overall health rating and CPET fc results, where patient’s age and sex were used as control variables.

Results:
With each score increase in the overall health rating, the odds of the fc value being normal than being mildly/moderately/severely reduced increases by 1.2 times, given the patient sex and age remain constant (95%CI (0.76,0.98), p = 0.019<0.05).

Conclusion:
The results reveal a statistically significant association between the patients' overall health rating and their CPET fc results. The higher the numerical value of the overall health rating and the better the patients feel about their health, the greater the probability that the patient will also have a better CPET fc result. Thus the CPET fc can be used as an objective value to assess a patient’s overall well being.
Abstract 330: Expression of Glycolytic Enzymes in Induced Pluripotent Stem Cells, Derived Megakaryocytes, and Endothelial Cells
Authors: Chandra Arun , Vaidya Dhananjay , Chang Rui , Knowles Josh , Carcamo-oribe Ivan , Kammers Kai , Martin Joshua , Taub Margaret , Hoyle Dixie , Gao Yongxing , Yanek Lisa , Kral Brian , Faraday Nauder , Becker Diane , Mathias Rasika , Cheng Linzhao , Leek Jeffrey , Wang Zack , Becker Lewis
Published: October 2020

Introduction:
Induced pluripotent stem cells (iPSC) have a significant capacity for self-renewal and can differentiate into any somatic cell type, such as megakaryocytes (MK) and vascular endothelial cells (EC). We have previously shown that iPSC-derived MKs (IPSC-MK) upregulate the gene expression levels of irreversible glycolytic enzymes committing glucose to glycolysis in comparison to their parental iPSC lines (Vaidya et al., 2016). This pattern differed from past comparisons between somatic source cells and iPSCs (Chung et al., 2007; Varum et al., 2011; Zhang et al., 2011). To further corroborate our data, we extended our analysis to iPSC-derived ECs (iPSC-EC).

Methods:
We studied expression of glycolytic enzyme genes in 30 human iPSC lines and paired iPSC-EC and iPSC-MK lines using RNA-seq. iPSC-EC to iPSC expression ratios for 11 glycolytic enzymes were estimated using a multilevel mixed-effect model regression, and a significance threshold of p < 0.05/11 = 0.0045 was used. A bivariate statistical analysis was used to assess the correlation between iPSC-EC and iPSC-MK data.

Results:
Of the 11 glycolytic enzymes studied, 4 were highly expressed in iPSC-ECs (Log2(iPSC-EC/iPSC) ≥ 1) while 1 was highly expressed in iPSCs (Log2(iPSC/iPSC-EC) ≥ 1). Transcripts for functionally irreversible enzymes including ADPGK, PFKP, and PKLR displayed higher expression levels in iPSC-ECs compared to iPSCs. Similarly, 6 glycolytic genes demonstrated higher expression in iPSC-MKs, while 2 were more highly expressed in iPSCs. The functionally irreversible enzymes HK1, ADPGK, and PFK exhibited higher expression levels in iPSC-MKs. The ratios of gene expression (derived-lines:iPSCs) for all 37 genes in the iPSC-MK results correlated with those of the iPSC-EC results: Pearson’s r = 0.52, p = 0.001.

Conclusion:
Both iPSC-ECs and iPSC-MKs upregulate the expression of glycolytic enzymes compared to their parental iPSCs. In light of data showing that differentiated cells are more metabolically active and have more mitochondria than iPSCs (Chandra et al., 2020), these results suggest that differentiation of iPSCs is accompanied by an increase in both glycolytic and TCA cycle activities, with glycolytic generation of pyruvate used for mitochondrial production of ATP.
Abstract 337: Cardiac Inflammation Contributes to Heart Failure With Preserved Ejection Fraction (HFpEF) in Diabetic Mice With Low Aldehyde Dehydrogenase 2 (aldh2) Activity
Authors: Pan Guodong , Palaniyandi Suresh S
Published: October 2020

Heart failure with preserved ejection fraction (HFpEF) is the prevalent form of cardiac complication in type 2 diabetes, which is associated with higher mortality. Inflammation plays a central role in the development of HFpEF, although the mechanism is unclear. Aldehyde dehydrogenase 2 (ALDH2), a mitochondrial enzyme, detoxifies oxidative-stress mediated 4-hydroxy-2-nonenal (4HNE) and thereby confers cardioprotective in diabetes. ALDH2*2 is the most common genetic mutation which causes a low ALDH2 activity. We hypothesize that low ALDH2 activity increases cardiac inflammation and consequentially aggravates HFpEF in diabetic mice. We bred a diabetic mouse model which carries mutant leptin receptor (same as db/db mice) and mutant ALDH2*2 (AL mice). The mice with double mutations are termed as ALDH2*2-fat (AF) mice. The ejection fraction (EF) is preserved in db/db (65.9±2.2%) and AF (67.2±3.9%) diabetic mice compared to db/m (68.5±4.6%, p>0.05 vs db/db) and AL (68.6±4.8%, p>0.05 vs AF) non-diabetic mice at 20 weeks. The running stress by treadmill decreased EF in AF mice (56.3±5.0%) compared to db/db (65.0±3.3%; p<0.05), db/m (70.5±2.8%; p<0.01) and AL (66.4±4.1%; p<0.05). The maximum running distance was significantly decreased in AF (128.7±28.8m) mice compared to db/db (193.2±14.2m; p<0.05), AL (305.2±21.5m; p<0.01) and db/m (371.3±16.6m; p<0.01) mice. In heart sections, the CD64+ macrophages in AF mice (88.64±22.12 /mm
2
) were significantly increased, compared to db/db (53.24±12.55 /mm
2
, p<0.05), db/m (12.57±2.56 /mm
2
, p<0.01) and AL mice (47.89±13.97/mm
2
, p<0.05). In conclusion, the low ALDH2 activity contributes to cardiac inflammation and thus HFpEF in diabetic mice.
Abstract 338: Limited Reduction of Heart Rate Promotes Cardiac Regeneration by Switching the Energy Metabolic Mode in Cardiomyocytes
Authors: Tan Jing , Yang Ming , Wang Haiping , Wu Yandi , Li Yuanlong , Deng Shijie , Huang Tongsheng , Shen Conghui , Li Xinghui , Cai Weibin
Published: October 2020

Aims:
Lack of cardiac regeneration with robust fibrosis response to the acute myocardial injury is the main obstacle to clinical treatment of cardiovascular diseases in humans. Stimulating the proliferation of endogenous cardiomyocytes (CMs) and replacing the scarred tissue with new functional CMs is a potential therapeutic strategy to the patients with heart failure. Heart rate reduction (HRR) is regarded as an effective clinical treatment for myocardial infarction. However, the mechanism of HRR promoting the recovery of cardiac function after injury still remains controversial, and whether there is any endogenous CM proliferation induced by HRR is undefined.

Methods and results:
The beating of CMs was reduced
in vitro
and heart rate (HR) of adult mice and different animal models of myocardial injury were modulated by six antiarrhythmic drugs to determine the role of HR in CM proliferation and cardiac repair. RNA-seq, extracellular flux analysis, metabolic flux analysis, and metabonomics were used to study the CM metabolism after HR modulation. We verified that reducing the beating can induce CM proliferation both
in vitro
and
in vivo
physiologically, and HRR also promoted cardiac regenerative repair after myocardial injury as well, reversely, increasing HR showed the opposite effect. Mechanistically, HRR reduced energy metabolism requirements and total ATP production of CMs but switched energy metabolic mode that the proportion of ATP production from aerobic glycolysis was increased, while from fatty acid oxidation was decreased. The switching of energy metabolic mode in CMs occurred in synchrony with the changes of glycolytic enzymes activities, these enzymes, including PFKFB3, PKM2, GAPDH, induced G1/S transition for cell cycle re-entry of CMs by upregulating the expression of cyclin D and CDK4/6 and facilitate substrates into the biomass needed to produce a new cell by biosynthesis. This coordinating function of glycolytic enzymes contributed to CM proliferation.

Conclusion:
Together, these results demonstrate that reduction of heart rate promotes CM proliferation by switching the energy metabolic mode, and highlight the potential therapeutic role of HRR in regenerative medicine.
Abstract 341: Protective Role of Myeloid Cell-specific Erbb3 Signaling After Transverse Aortic Constriction
Authors: Yin Haifeng , Lessard Amanda J , deKay Joanne T , Herrmann Yodit , Robich Michael P , Sawyer Douglas B , Ryzhov Sergey
Published: October 2020

Introduction:
Understanding molecular mechanisms underlying cardiac hypertrophy is crucial for protecting against cardiac remodeling, or slowing down its destined pathway to heart failure. Recent studies revealed that myeloid cells play an important role in the regulation of hypertrophic remodeling. Neuregulin-1 (NRG-1) has been shown to play an essential role in the regulation of tissue-protective and pro-survival processes in response to tissue injury in the cardiovascular system. We find that NRG-1 receptors, including ErbB2 and ErbB3, are expressed and functionally active in cardiac myeloid cells. We hypothesized that the NRG-1/ErbB3 signaling in myeloid cells plays a protective role in cardiac hypertrophic remodeling. To test this hypothesis, we examined the effect of
Erbb3
gene ablation in mouse myeloid cells on cardiac hypertrophic remodeling induced by pressure overload.

Methods and results:
Myeloid-specific ErbB3-deficient mice (ErbB3
MyeKO
) were generated by crossing
Erbb3
-floxed mice with LysM-Cre transgenic mice. Cardiac hypertrophic remodeling was established in mice by transverse aortic constriction (TAC). Five days after TAC, survival was dramatically reduced in male ErbB3
MyeKO
mice (20% vs. 90%,
p=0.021
, MyeKO vs. MyeWT). The post-mortem examination of lung weight to body weight ratio suggested that acute pulmonary edema was developed in ERBB3
MyeKO
mice. In order to determine the cellular and molecular mechanisms involved in the increased mortality in ErbB3
MyeKO
males, cardiac cell populations were examined at day 3 post-TAC using flow cytometry. A significant accumulation of myeloid cells was found in control but not in ErbB3
MyeKO
males. This was accompanied by increased proliferation of Sca-1 positive non-immune cells (endothelial cells and cardiac progenitors) in control but not ErbB3
MyeKO
males. An antibody-based protein array analysis revealed that IGF-1 expression was significantly downregulated only in ErbB3
MyeKO
males after TAC.

Conclusion:
Our data highlight the important role of myeloid cell-specific ErbB3 signaling in the accumulation of myeloid cells that contributes to the development of compensatory hypertrophy and the prevention of acute heart failure in male mice.
Abstract 343: Glycogen Synthase Kinase 3β Localizes to the Cardiac Myofilament via Py216 and Dynamically Modulates Calcium Sensitivity
Authors: Stachowski Marisa , Papadaki Maria , Zlobin Andrei , Martin Thomas , Aima Muntu Nitha , Martin Jody , Moravec Christine S , Kirk Jonathan A
Published: October 2020

Localization of kinases to sub-cellular compartments allows them dynamic control over specific subsets of their targets. We previously found GSK-3β could modulate myofilament Ca
2+
sensitivity. However, whether GSK-3β modulates Ca
2+
sensitivity
in vivo
, if it localizes to the myofilament, and the consequences, are unknown. In myofilament enriched LV tissue from human non-failing and heart failure (HF) patients (n = 66) we found GSK-3β does localize to the myofilament and is altered by sex, age, and HF. To determine its
in vivo
functional role, we used myocyte specific inducible GSK-3β knock-out mice for skinned myocyte force-calcium experiments and found that GSK-3β reduction reduced calcium sensitivity. Further, we measured function in human samples and found myofilament GSK-3β levels directly correlated to Ca
2+
sensitivity. To establish
how
GSK-3β binds to the myofilament, we performed co-IP and IHC with phosphorylated forms of GSK-3β. GSK-3β phosphorylated at Y216 had a high affinity for the myofilament and localized to the z-disc. Mutating Y216 to a phospho-mimetic increased binding to the myofilament while mutating it to a phospho-null ablated binding.To identify GSK-3β’s myofilament targets, we performed mass spectrometry on myofilament phospho-enriched samples from GSK-3β KO and WT mice. As GSK-3β modulated Ca
2+
sensitivity, we expected to detect thin filament proteins. However, in agreement with its localization to the z-disc, GSK-3β primarily phosphorylated z-disc proteins.In the GSK-3β KO mice, strain analysis revealed the posterior wall contracted significantly earlier than the anterior wall, indicating baseline mechanical dyssynchrony. The WT mice had synchronous contraction, and interestingly there was significantly higher myofilament GSK-3β in the anterior wall compared to the posterior wall, a difference we hypothesize maintains synchrony. Thus, losing this fine control over Ca
2+
sensitivity as in the GSK-3β KO mice would induce mechanical dyssynchrony. Overall, these findings reveal that GSK-3β dynamically localizes to the myofilament to modulate Ca
2+
sensitivity through Y216 phosphorylation. The consequence of this “fine tuning” maintains chamber level mechanical synchrony.
Abstract 346: Cardiac Expression of Fetal and Adult SCN5A Isoforms in Sudden Unexplained Infant Death (SUID)
Authors: Williams Alexandra F , Tomasek Kelsey , Gregory Kyle , Fulmer Carlos , Bozeman Cole , Bryan Audra F , Li Feng , Kannankeril Prince J , Su Yan Ru
Published: October 2020

Sodium Voltage-Gated Channel Alpha Subunit 5 (SNC5A) plays a vital role in cardiac depolarization and has been implicated in Long QT Syndrome and sudden cardiac death.
SCN5A
encodes fetal and adult isoforms, differing by splicing of exon 6A or 6B, respectively. Fetal SCN5A decreases peak sodium current, leading to slower channel activation and inactivation and providing a potential substrate for arrhythmia. Little is known about developmental expression of these isoforms. We investigated developmental expression of SCN5A isoforms in infants, toddlers, and adults, including expression ratios in SUID cases and controls (0-12 months). We extracted RNA from heart tissue collected at autopsy (fetus-24 months) with 80 SUID cases and 27 controls, and 5 adult donor hearts. RNA Integrity Numbers ranged from 4.8-9.5. Relative SCN5A isoform expression was quantified by qRT-PCR. Non-parametric t-test and ANOVA were used; P<0.05 was considered statistically significant. Our results show a significant stepwise increase in adult/fetal isoform expression with age. Relative expression of adult/fetal SCN5A increased significantly from fetal-4 months to 5-24 months (P<0.0001), and again in adulthood (P<0.0001). There was no significant difference between case and control groups at 0-4 or 0-12 months. Expression of adult/fetal SCN5A increases significantly from birth to 24 months of age and into adulthood. Relative adult/fetal SCN5A expression is lowest during the neonatal period through 4 months, corresponding with the peak incidence of SUID. Further investigation of protein expression of SCN5A isoforms may shed light onto the age-dependent incidence of sudden death.
Abstract 351: Focal Adhesion Kinase Promotes Tissue Destruction in Aortic Dissection in Mice
Authors: Majima Ryohei , Aoki Hiroki , Nakao Eichi , Hashimoto Yohei , Ito Sohei , Hayashi Makiko , Ohno-urabe Satoko , Frusho Aya , Nishida Norifumi , Hirakata Saki , Fukumoto Yoshihiro
Published: October 2020

Aortic dissection (AD) is a fatal disease due to the sudden destruction of the aortic medial layer. Currently, Molecular pathogenesis of AD is unknown. We investigated the role of focal adhesion kinase (FAK), a mechanosensitive signal transducer, in AD pathogenesis. We created a mouse model of AD with a continuous infusion of beta-aminopropionitrile (150 mg/kg/day), a collagen crosslink inhibitor, and angiotensin II (1,000 ng/kg/min) (BAPN + Ang II) by osmotic pumps. This AD model showed about 60% mortality within 2 weeks due to AD rupture. Immunohistochemical staining for activated FAK revealed that FAK was inactive in normal mouse aorta, but was strongly activated in the aortic walls after BAPN + Ang II infusion. Immunofluorescence staining showed that FAK was activated mainly in smooth muscle cells after the BAPN + Ang II challenge. Western blot analysis revealed that FAK was activated in 3 days after BAPN + Ang II infusion before AD development, followed by transient reduction at day 7, and re-activation after AD development at day 14. We examined the effect of PND-1186, an orally available FAK inhibitor, on the severity as determined by the AD lesion length and the mortality of AD. Mice were administered with either vehicle or PND-1186 (150 mg/kg twice daily by oral gavage) during the BAPN + Ang II challenge (n=20 for each group). Administration of PND-1186 resulted in significant reduction in the lesion length of AD (vehicle; 12.5 ± 1.65 mm, PND; 7.46 ± 1.88 mm, P<0.05). The suppressive effect of PND-1186 was most significant in the aortic arch (vehicle; 2.13 ± 0.29 mm, PND; 0.85 ± 0.22 mm, P<0.01). Furthermore, PND-1186 significantly improved the survival rate of mice from 40.0% to 80.0% (P<0.01). Transcriptome analysis indicated that destruction and inflammation of tissue were suppressed by PND-1186 administration. These findings indicated that FAK plays an important role in AD pathogenesis, possibly by transducing the pathological stress to the tissue destructive response in the aortic walls. We propose that FAK is a potential therapeutic target to limit the fatal destruction of aortic walls in AD.
Abstract 369: Infarct Macrophage Secretome Factors Regulate Neutrophil Degranulation
Authors: Daseke Michael , Kalusche William , Flynn Elizabeth , Konfrst Shelby , Lindsey Merry L
Published: October 2020

Following myocardial infarction (MI), the left ventricle undergoes wound healing that begins withan intense inflammatory response to recruit leukocytes to the infarcted region. Infiltratedneutrophils degranulate, releasing a series of proteases including matrix metalloproteinase(MMP)-9 into the extracellular space. Macrophages also infiltrate into the infarct region, and wehypothesized that the macrophage may secrete factors that regulate neutrophil degranulation.Stimulating bone marrow derived neutrophils from C57BL/6J mice (3-6 month old male) withphorbol myristate acetate (PMA) induced degranulation, as evidenced by release of MMP-9.Co-stimulation with MI day 1 macrophage secretome (10% by volume) reduced PMA-induceddegranulation of MMP-9 by 8-fold (p<0.0014). Transcriptomic analysis of day 1 MI macrophagesrevealed galectin-3, vimentin, fibronectin, and murinoglobulin-1 were highly expressed.Examination of the day 1 MI macrophage secretome also showed high expression of vimentinand fibronectin. To further unmask signaling connections, neutrophils were co-stimulated withPMA and day 1 MI macrophage secretome, along with blocking antibodies for the 4 proteinsindividually. Galectin-3 inhibition promoted the macrophage secretome effect, whilemurinoglobulin-1 inhibition reversed the macrophage secretome effect. Neutrophil stimulationwith recombinant galectin-3 showed no additive effect with PMA on MMP-9 release, suggestingthat galectin-3 works through the protein kinase C signaling pathway to induce neutrophildegranulation. Neither Vimentin inhibition nor stimulation had an effect on neutrophildegranulation Overall, our results uncovered a role for galectin-3 and fibronectin in inducingneutrophil degranulation while murinoglobulin-1 may be a potent inhibitor of degranulation. Theday 1 MI macrophage therefore secretes both factors that promote as well as factors thattemper neutrophil degranulation. The balance of these factors determines the net effect of themacrophage secretome on neutrophil degranulation.
Abstract 373: Impacts of Harvesting Sites on Cardiac Extracellular Matrix Hydrogels Properties
Authors: Mulvany Emily , McMahan Sara , Xu Jiazhu , Yazdani Narges , Willits Rebecca , Liao Jun , Zhang Ge , Hong Yi
Published: October 2020

Introduction:
Cardiac extracellular matrix (cECM) hydrogel has been explored to treat myocardial infarction (MI). It possesses organ specific microenvironmental cues for resident cells and can be minimally invasive delivered into the heart using a transendocardial catheter. Recent clinical studies demonstrated the safety and feasibility of using cECM for treating MI patients. However, little is known about whether the region of cardiac tissue harvesting is critical for downstream applications. We performed
in vitro
studies to compare cECM hydrogels derived from porcine whole heart (WH), left ventricle (LV), and right ventricle (RV).

Materials and Methods:
Adult porcine hearts were obtained from a local USDA approved abattoir. The tissues were separated into 3 major categories: LV, RV, and WH tissues. Following our published protocol, tissues were decellularized and solubilized. The complete decellularization was confirmed by H&E staining and DNA content measurement. Chemical composition was examined using SDS-PAGE electrophoresis as well as Collagen and GAGs measurements. The gelation time, SEM (hydrogel microstructures), and mechanical properties (storage and complex modulus) were studied. Human cardiomyocytes (CMs) and mesenchymal stem cells (MSCs) were separately seeded on the hydrogels to examine their responses. Cell viability and vascular differentiation of human MSCs were examined.

Results and Discussion:
The cECM from WH has more complex chemical composition compared with cECM from LV and RV. All three types of cECM hydrogels share many similarities in terms of their microstructure, gelation time and mechanical properties. WH-derived cECM hydrogels have larger variations in storage modulus (G’) and complex modulus (G*) compared with the other two types of cECM hydrogels. Both human CMs and MSCs could maintain high cell viability on all hydrogels. Regardless of harvesting sites, all cECM hydrogels have equivalent potency in directing vascular differentiation of seeded human MSCs.

Conclusions:
The cECM hydrogels from WH, LV and RV exhibited similarity in material properties and cell response
in vitro
. Future fabrication of cECM hydrogels from WH would increase the yield and decrease production cost.
Abstract 375: Insights Into Molecular Mechanism of Cardiovascular Disorder in HIV+ Patients
Authors: Gupta Manish , Scanlon Kaylyn , Mukker Avni , Gupta Deepti , Rappaport Jay , Gupta Manish K
Published: October 2020

Background:
Antiretroviral therapy (ART) improves the survival of people living with HIV (PLHIV); however, the rate of cardiovascular disorder and heart failure is significantly increased among the PLHIV. Molecular basis of heart failure in the PLHIV undergoing antiretroviral drug treatment is not clear. The aim of this study is to explore the role of antiretroviral drugs in post translational modification of histones and its epigenetic regulation of gene expression in cardiomyocytes.

Methods and Results:
Primary rat ventricular cardiomyocytes were treated with a combination of antiretroviral drugs (5 μM of Atazanavir, Abacavir, Ritonavir and Lamivudine) for 4, 12 and 24 hours, and expression of major histone marks playing a role in gene activation (H3K9ac and H3K27ac) and repression (H3K27me3, H3K9me3) were evaluated by western blotting. Our data suggest that treatment with antiretroviral drugs leads to de-acetylation at H3K9ac and H3K27ac, and promotes methylation at H3K27me3 and H3k9me3. Additionally, the expression of epigenetic modifying enzymes was examined by PCR array in cardiomyocytes treated with antiretroviral drugs. PCR array data show that histone deacetylase enzyme Sirt1/2, and methyltransferase enzyme Suv39h1 and Ezh12 were upregulated in drug treated cardiomyocytes. Further, western blot data show that Sirt1, Suv39h1 and Ezh2 protein expression was significantly upregulated in drugs treated cardiomyocytes. Moreover, expression analysis of human cardiac tissue further shows that expression of Sirt1, Suv39h1 and Ezh2 was significantly upregulated in HIV+ patients heart compares to healthy donor. Mechanistically, our data show that expression of epigenetic modifying enzymes was differentially regulated in drug treated cardiomyocytes which may lead to epigenetic modifications of histone proteins.

Conclusion:
Antiretroviral drug treatment promotes epigenetic alteration in the chromatin which may lead to a change in gene expression of cardiomyocytes. This study may lead to novel therapeutic strategies for the treatment of heart failure in PLWHA.
Abstract 377: The Role of Impaired Lymphatic Drainage in Cardiac Transplantation
Authors: Ginn Sydney C , Wang Lanfang , Levit Rebecca D
Published: October 2020

Functional lymphatic drainage inherently modulates cardiac function by maintaining the immune response and tissue-fluid homeostasis. During cardiac transplantation, the lymphatic collecting vessels are severed at the time of heart excision and not surgically reconstructed in the recipient. The consequence resulting from impaired lymphatic drainage in transplanted hearts is unknown. We hypothesize disruption of lymphatic drainage potentiates chronic inflammation by impeding the egress of immune cells and pro-inflammatory cytokines out of the myocardium exacerbating transplant rejection.

Methods:
Banked human allograft biopsies were utilized to retrospectively evaluate lymphatic differences between patients that did and did not develop chronic transplant rejection from 1 week to 5 years after surgery. Immunofluorescence staining permitted quantification of normalized lymphatic vessel number and area throughout the lifespan of each cardiac allograft (n=24). Autopsy patients with non-cardiac related fatalities served as controls to delineate normal cardiac lymphatic distribution (n=6).

Results:
Patients without chronic rejection displayed an initial presence of lymphatic vasculature that steadily declined (n=12), while patients with chronic rejection exhibited a delayed increase in lymphatic development (n=12).

Conclusions:
These data show significant differences in lymphatic area between patients with and without chronic transplant rejection at critical timepoints, suggesting delayed lymphangiogenesis may correlate with rejection.

Translational Impact:
These preliminary human data support further investigation into lymphatic-modifying therapeutics to prolong the life of cardiac allografts.
Abstract 378: Physiologic Role of
Rpl13a
SnoRNAs in Cell Size Regulation
Authors: Ho Hsiang Ting T , Holley Christopher L
Published: October 2020

Objectives:
Box C/D small nucleolar RNAs (snoRNA) are a multifunctional family of ncRNAs that play a critical role in guiding 2'-
O
-methylation (Nm) of ribosomal RNA (rRNA). My work has recently revealed that box C/D snoRNAs can also direct methylation of messenger RNA (mRNA), and that this methylation can regulate mRNA translation in the heart. In studies of genetically-engineered mice lacking four box C/D snoRNAs, I have observed that the knockout animals have relatively small hearts compared to wild-type. The objective of my current study is to define the specific mechanisms by which box C/D snoRNAs regulate heart size.

Methods:
I investigated the hearts of genetically-engineered mice lacking four box C/D snoRNAs from the
Rpl13a
locus (snoRNAs
U32a, U33, U34
and
U35a
). I also used antisense oligonucleotides to knock down these snoRNAs in H9c2 rat cardiomyoblasts. Changes in gene and protein expression were assessed by qPCR and immunoblot. Relative Nm modification of mRNA was determined by reverse transcription at low dNTP concentrations, followed by real-time PCR (RTL-P).

Results:
Germline knockout of the four box C/D
Rpl13a
snoRNAs (
U32a, U33, U34
and
U35a
) in adolescent mice produces developmentally smaller hearts. In H9c2 rat cardiomyoblasts, knockdown of
Rpl13a
snoRNAs by antisense oligonucleotides significantly reduces H9c2 cell size. These concordant effects on organ and cell size suggest that the
Rpl13a
snoRNAs might be regulating critical pathways that determine cell growth. Using a candidate gene approach, I found that
Mtor
mRNA and protein expression is significantly reduced in hearts from
Rpl13a
snoRNA knockout mice. Preliminary results using the RTL-P method suggest that
Mtor
is subject to snoRNA-guided Nm modification, which is decreased in
Rpl13a
snoRNA knockout mouse hearts.

Conclusion:
These results suggest that
Rpl13a
snoRNAs regulate cardiomyocyte growth, at least in part, by guiding Nm modification of
Mtor
mRNA.
Abstract 379: Metformin Induces Energetic Reprogramming in Human Cardiomyocytes
Authors: Emelyanova Larisa , Yan Yasheng , Kroboth Stacie , Warner Catherine , Kress David , Rizvi Farhan , Bai Xiaowen , Jahangir Arshad
Published: October 2020

Introduction:
Metformin (Metf) is an oral hypoglycemic agent widely used to treat type 2 diabetes. It also lessens myocardial injury and reduces cardiovascular mortality. However, the exact molecular mechanisms of Metf therapeutic action in the human heart remain obscure. The aim of this study was to evaluate the cardioprotective effect of Metf on cardiomyocytes derived from human-induced pluripotent stem cells (hiPSC-CMs).

Methods:
The hiPSC-CMs were derived from an hiPSC line generated from human dermal fibroblasts and treated with Metf (0-20 mM) for 24 hours. Oxygen consumption rate (OCR) was measured using XF96 Extracellular Flux Analyzer. Metabolites were profiled using a liquid chromatography coupled to tandem mass spectrometry. Tukey’s pairwise comparison and one-way ANOVA were applied for comparison between groups. P < 0.05 was considered significant.

Results:
At concentrations ≤ 2.5 mM, Metf significantly enhanced the cellular OCR (Fig. A) and enhanced mitochondrial biogenesis (Fig. B) via AMPK signaling (Fig. C). This effect was abrogated by compound C, an inhibitor of AMPK. At concentrations > 5 mM, Metf reduced the cellular OCR and activated glycolysis (Fig. D) and glutaminolysis (Fig. E) in the cardiomyocytes.

Conclusion:
Thus, in the human heart, Metf might improve cardioprotection owing to its dual effect on mitochondria. At low doses, it activates mitochondrial biogenesis via AMPK signaling and increases the OCR. At high doses, it causes metabolic reprogramming by enhancing glycolysis and glutaminolysis as a compensatory mechanism in response to inhibition of the mitochondrial oxidative phosphorylation.
Abstract 383: Cavβ2a Transgenic Mouse as a Hfpef Model With Hypercontractile Cardiomyocytes
Authors: Li Yijia , Zhang Xiaoying , Jin Jianguo , Molkentin Jeffery , Houser Steven , Chen Xiongwen
Published: October 2020

Objective:
Heart failure (HF) with preserved ejection fraction (HFpEF) is characterized by a preserved cardiac EF, the presence of HF symptoms and diastolic dysfunction. There is a lack of animal models for exploring the mechanisms and treatments of HFpEF. This study aimed to test if cardiomyocyte (CMs) specific, inducible Cavβ2a transgenic (Cavβ2a TG) mice, having hypercontractile CMs, could be a model for HFpEF.

Methods:
High (HE) and low (LE) expression Cavβ2a TG mice were studied since transgene expression at the age of 2m till the age of 8m monthly to evaluate the systolic and diastolic function. At 8m, animals were euthanized for intra-left ventricular hemodynamic measurement, myocyte function and histological analyses, and Western blotting measurements.

Results:
LE and HE TG ventricular myocytes (VMs) had greater Ca2+ currents at the age of 2m (LE increased by 95.5%; HE increased by 171.9% ) and maintained at a similar level at the age of 8m. VM contraction and calcium transients had greater amplitudes in TG than in control VMs while the time from the peak contraction to 90% relaxation and the tau of Ca2+ transient decay of VMs were not different between groups probably due to enhanced NCX expression and increased SERCA activity because of increased phospholamban phosphorylation at the Thr17 site. Till 8m, LE and HE mice had a higher level of mortalities than control mice (LE: 32%, HE: 15%, control:5%), but more HE (87.5%) mice showed pleural effusion. The EF was highest in LE mice at the age of 2m but decreased to the same (>60%) as in HE and control mice at 8m. The ratio of mitral E to mitral inflow A wave velocities (E/A) was increased significantly at 3 and 4m then decreased to the lowest in HE at 5m followed by a rise at 6-8m, while LE mice had the lowest E/A at 5m and 6m and increased at 8m. E wave to mitral annulus e’ velocity (e’ wave) was higher in LE and HE groups than control mice. More fibrosis of the LV tissue, cardiomyocyte necrosis was observed in HE than in LE hearts. LE and HE mice had greater left ventricular wall relaxation rates, as indicated by larger reversed radial and longitudinal strain rates.

Conclusion:
LE and HE mice showed progression of diastolic dysfunction, from impaired relaxation to restrictive filling pattern. Cavβ2a TG mouse is a model for HFpEF for exploring HF pathobiology and mechanisms.