bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 423: Cardiomyocyte Maturation and Multinucleation in Postnatal Swine
Authors: Velayutham Nivedhitha , Alfieri Christina M , Agnew Emma J , Riggs Kyle W , Baker Richard S , Zafar Farhan , Yutzey Katherine E
Published: October 2019

Objectives:
Cardiomyocyte (CM) cell cycle arrest and decline of mononucleated diploid CMs have been implicated in loss of regenerative potential in postnatal mouse hearts. Sarcomeric and extracellular matrix (ECM) maturation also occur concurrently in mice, influencing CM proliferative arrest. Recent studies show a 3-day neonatal period of cardiac regeneration in pigs similar to mice, but the dynamics of postnatal pig CM growth are unknown. Our objective is to explore cardiac cell cycling, growth and maturation in postnatal pigs to understand the events guiding loss of cardiac regenerative capacity in large mammals.

Methods & Results:
Left-ventricular tissue from farm pigs (White Yorkshire-Landrace) at Postnatal day (P)0, P7, P15, P30, 2 months (2mo) and 6mo were utilized. CM dissociations revealed predominant CM mononucleation at birth in swine, with persistence of ~50% (1186/2537 cells, n=5) mononucleated CMs at P15, and ~10% (227/1785 cells, n=4) at 2mo. By 6mo, pig CMs are entirely multinucleated, exhibiting 4-16 nuclei per cell. Assessing hypertrophic growth in dissociated pig CMs revealed longitudinal CM growth relative to increased nucleation at all ages. However, onset of diametric hypertrophy only occurs beyond 2mo. When nuclear pHH3 and mRNA expression of cell cycle genes was assessed, pig hearts show robust cell-cycling up to 2mo. Also, fetal TNNI1 and MYH6 are active up to 2mo in pig hearts. Ongoing studies on collagen remodeling indicate ECM remodeling in swine occurs beyond P7. Future studies are designed to identify nuclear ploidy in postnatal pig CMs and measure cytokinesis.

Conclusions:
Cardiac maturational events are staggered over a 2-6 month postnatal window in pigs, with older pig hearts exhibiting extensive CM multinucleation and differences in longitudinal versus diametric CM growth. These fundamental variations in CM growth characteristics are important to consider when designing preclinical trials for cardiac regenerative strategies in pigs. Also, despite a similar period of regenerative capacity as mice, pig hearts do not undergo loss of CM cell cycling and mononucleation until 2mo after birth. Utilizing pigs may thus offer unique opportunities to study aspects of heart regeneration unavailable in other animal models.
Abstract 432: Rapamycin Can Ameliorate Antiretroviral Drug Mediated Cardiotoxicity
Authors: Alapati Spoorthi , Gupta Manish K
Published: October 2019

Introduction:
In current therapy, due to application of combined antiretroviral drugs (cART), HIV related mortality significantly reduced, and AIDS became a chronic disease. Although, cART treatment increases life expectancy of AIDS patients, growing AIDS population suffer due to several organ diseases and heart failure is one of the leading causes of comorbidity and mortality.

Hypothesis:
We tested the hypothesis that application of cART induces cardiotoxicity through inhibition of cellular protein quality control (PQC) and dysregulation of mitochondrial function.

Method and results:
Cellular protein quality control was analyzed using cardiac tissue samples from HIV+ patients treated with cART and healthy donors. Western blot shows that there is upregulation of autophagy marker protein LC3 II in ART treated heart compared to healthy donor. Additionally, we found that there are abnormalities in the mitochondrial oxidative phosphorylation complexes (OXOPHOS) in the ART treated patient’s heart, compared to healthy donor heart. Using neonatal rat ventricular cardiomyocytes, we tested the role of ART in cellular PQC and mitochondrial function. Our data shows that cardiomyocytes treated with the cART (Ritonavir+ Atazanavir+ abacavir+ Lamivudine) have increased expression of autophagy proteins LC3 II and P62 and higher level of pELF2α. These suggest that, the cardiomyocytes treated with ART have dysregulation of autophagy and increased ER stress. Further, we found that cardiomyocytes treated with cART have high level of mitochondrial superoxide and decreased mitochondrial membrane potential. Mitochondrial function detected in whole cells shows that in ART treated cardiomyocytes there is significantly low OCR compared to control cells. Interestingly, application of pharmacological drug rapamycin can improve the cellular PQC and mitochondrial function in ART treated cells.

Conclusion:
ART treatment induces cardiotoxicity through inhibition of cellular PQC and dysregulation of mitochondrial function and rapamycin might be important therapeutic drug to reduce the ART mediated cardiotoxicity.
Abstract 435: SUMOylation Site in MCL-1 Regulates its Anti-Apoptotic Activity
Authors: Leon Leonardo J , Moyzis Alexandra G , Gustafsson Asa B
Published: October 2019

Myeloid Cell Leukemia-1 (MCL-1) is an anti-apoptotic BCL-2 protein upregulated in various types of cancer. It is also highly expressed in the myocardium. We previously found that MCL-1 plays a key role in maintaining mitochondrial homeostasis and myocyte survival. Cardiac specific deletion of MCL-1 in myocytes leads to mitochondrial dysfunction, loss of myocytes, and rapid development of heart failure. However, MCL-1’s function in the heart is unclear and the exact mechanism by which MCL-1 protects against apoptotic stimuli is not fully understood. Studies have reported that MCL-1 is subjected to phosphorylation and ubiquitination at several different residues which leads to changes in its stability. MCL-1 has a short half-life and proteasomal-mediated degradation of MCL-1 allows for apoptosis to proceed. Here, we screened the amino acid sequence of MCL-1 to identify novel sites of post-translational modifications involved in regulating MCL-1 function. We discovered the presence of a sumoylation site at lysine 219 that is conserved between species. To examine the function of the site, we generated a mutant of MCL-1 where the lysine residue was mutated to an arginine, MCL-1K219R. First, we investigated whether the sumoylation site was involved in regulating protein stability of MCL-1, but we found no differences in the degradation rates between MCL-1 and MCL-1K219R at baseline and after staurosporine treatment. Interestingly, MCL-1K219R failed to protect against staurosporine-induced apoptosis, while both MCL-1 and MCL-1K219R protected against doxorubicin-mediated cell death. This suggest that the mechanism of doxorubicin-mediated cell death is different from staurosporine. Moreover, MCL-1 has been reported to prevent activation of apoptosis via the mitochondrial pathway by sequestering pro-apoptotic BCL-2 proteins such as PUMA and NOXA. Hence, we examined whether MCL-1 sumoylation is involved in regulating its interaction with pro-apoptotic proteins. A better understanding of how MCL-1 regulates cell survival will not only aid the development of drugs that combat harmful cells, but will also help the production of drugs that effectively protect limited and valuable cells like myocytes.
Abstract 441: The Role of Brown Adipose Tissue in Aging-Induced Cardiovascular Impairments
Authors: Abay Eaman , Mansour Megan M , Peres Valgas Da Silva Carmem , Baer Lisa A , Shettigar Vikram , Stanford Kristin I , Ziolo Mark T
Published: October 2019

Adults age 65 and older are the fastest-growing demographic in the United States population and pose a significant healthcare challenge. Among several other physiological changes, aging is notably associated with an increased risk of developing cardiovascular disease. Aging leads to a decrease in brown adipose tissue (BAT), a thermogenic tissue rich in mitochondria that converts chemical energy into thermal energy. Importantly, BAT plays a role in combatting metabolic and cardiovascular impairments. Thus, while it is known that aging is associated with both impaired cardiovascular function as well as decreased BAT mass, the specific role of BAT in age-induced impairments in cardiac function remains unknown. For this study, we utilized an old C57BL/6 mouse population (18 months of age) that was divided to sham-operated (control), +BAT (mice surgically transplanted with BAT), and -BAT (or BATless; mice in which BAT was surgically removed) groups. Echocardiography was performed before (baseline) and 3 months after surgery (BAT transplantation or removal). Our data show that mice transplanted with BAT had enhanced cardiac function compared to control, suggesting that increasing BAT mass can reverse age-induced cardiac dysfunction. Moreover, removal of BAT not only resulted in a further deterioration of cardiac function but also adverse remodeling, suggesting that a decrease in BAT is detrimental to the heart. In conclusion, the decrease in BAT as we age plays a significant role in the cardiac dysfunction with senescence and that increasing BAT mass may be a novel therapeutic approach.
Abstract 444: The Phase of Third Harmonic of Radial Pulse Predicts Cardiac Risk in Asymptomatic Patients With Type 2 Diabetes
Authors: Chang Chi-Wei , Liao Kuo-meng , Chang Yi-Ting , Wang Sheng-Hung , Chen Ying-chun , Wang Gin-Chung
Published: October 2019

Patients with type 2 diabetes (T2DM) have a significantly increased risk of heart disease and adverse cardiac events(ACE). Many of asymptomatic patients with T2DM have myocardial perfusion defects or coronary artery disease and are not recognized by traditional risk factors. The third harmonic of radial pulse has been proved a risk factor for myocardial ischemia. We sought to further investigate if the third harmonic phase of the radial pulse(P3) could be an independent predictor of ACE and cardiac dysfunction in asymptomatic T2DM patients. The study consisted of 1968 asymptomatic T2DM patients who had no history of angina and cardiovascular disease. We conducted the pulse wave measurement at baseline and dived the enrolled patients into quartile groups based on P3 value(>3.2, 3.0 to 3.2, 2.7 to 3.0, and <2.7). Participated patients received an average of 1.8 years of follow-up. To assess the risk of ACE , the primary outcomes were the composite of myocardial infarction, heart failure, and cardiovascular death. To measure the risk of cardiac dysfunction, the secondary outcomes were composed of newly discovered single or multi coronary stenosis, myocardial ischemia, and left ventricular dysfunciton. Cox proportional hazards model were used. Reference to patients with P3>3.2, the hazard ratios(HR) of patients with P3<2.7 were as follows: (1) myocardial infarction(HR, 1.98; 95% CI, 1.32-2.98), heart failure(HR, 1.64; 95% CI, 0.88-3.06), and cardiovascular death(HR, 3.51; 95% CI, 0.73-16.9); (2) Single coronary stenosis(HR, 10.1; 95% CI, 1.29-78.7), multi coronary stenosis(HR, 9.06; 95% CI, 1.15-71.5), myocardial ischemia(HR, 3.05; 95% CI, 1.37-6.80), and low left ventricular ejection fraction(HR, 2.09; 95% CI, 1.13-38.7). The trend analysis further demonstrated that the reducing P3 leads to higher incidence of both primary and secondary composite outcomes(P<0.001). After adjusting for age, gender, smoking, systolic blood pressure, dyslipidemia, duration of diabetes, and Hba1c, P3 was still an independent predictor of ACE and cardiac dysfunction (P
trend
<0.005). Conclusively, this report suggested that P3 provides independent predictive value for risk assessment of ACE and cardiac dysfunction in asymptomatic patients with T2DM.
Abstract 447: Development of CD47-nanomedicine as Novel Treatment for Specific Mitigation of Thrombospondin 1-Induced Vascular Dysfunction
Authors: Yao Mingyi , Ganguly Samayita , Ebrahimi Aren , Elbayoumi Tamer
Published: October 2019

The trans-membrane receptor CD47 is considered a central relay of thrombospondin 1 (TSP-1) mediated responses in human cells/tissues, and ubiquitously serves as a marker of self on hematopoietic cells.

Preliminary studies of TSP-1 binding with soluble human CD-47, serving as decoy recombinant protein (rhCD47p), demonstrated molar ratio ≥ 3 folds of rhCD47p abolished all TSP1-vascular CD47 receptor signaling. Accordingly, we attached multiple rh-CD47p ligands onto liposomes (NanoLip), as clinical prototype therapy. Novel rh-CD47p-NanoLip can avoid blood mononuclear cells, while binding many circulating TSP1 molecules, thus eliminating excessive plasma TSP-1, often implicated in pulmonary arterial hypertension (PAH) and ischemia-reperfusion injuries.

Direct coupling of rh-CD47p, onto NanoLip surface was achieved using a sulfo-NHS-reaction between amino groups on rh-CD47p and corbodiimide-activated NanoLip, followed by column purification and physico-chemical analysis. Pharmaceutically, rh-CD47p-NanoLip formulation (av. size = 86±6.0nm) engaged 67±11μg/mL of protein, while retaining 86% of native rh-CD47p activity. Western blots and TSP-1-ELISA revealed dosimetric binding of tirmeric human TSP-1: rh-CD47p-NanoLip. Activated macrophages showed substantial decrease in phagocytosis of labeled rh-CD47p-NanoLip, compared to plain- and IgG-NanoLip controls (5- and 4- folds, respectively), indicating self-marker function. Conversely, binding of exogenous TSP-1 caused significant upturn of internalized rh-CD47p-NanoLip (p<0.05, n=4), due to TSP-1/nano-complex formation. Therapeutically, in isolated mouse thoracic aorta model, mimicking high plasma levels of TSP1 in PAH patients, titrated doses of CD47p-NanoLip neutralized TSP1-impaired vasodilation, back to the same level as controls (p<0.001, vs. TSP-1, n=4).

This is the first report of its kind about CD47-based immunomodulatory nano-therapy specifically targeting TSP-1. The extracellular domains of native CD47 receptors of TSP1 were successfully “nano-modified” into a candidate systemic decoy pharmaceutical, rh-CD47p-NanoLip, to scavenge and eliminate pathologically elevated TSP-1 plasma levels, to abrogate TSP-1-associated vascular complications.
Abstract 449: Transplantation Alters Function and Clonality of Cytomegalovirus-Responsive T Cells
Authors: Higdon Lauren E , Schaffert Steven , Saligrama Naresha , Davis Mark M , Khatri Purvesh , Maltzman Jonathan S
Published: October 2019

Background:
Cytomegalovirus (CMV) infection is a major complication of organ transplantation. CMV establishes lifelong latency in infected individuals. Healthy people are protected from CMV reactivation by immune memory, but immunosuppression limits protection in transplant recipients. Despite antiviral prophylaxis strategies, long-term survival is impaired. CMV-responsive memory CD8 T cells expand during the first year after transplantation in the absence of detectable viremia. The observed expansion correlates with protection against CMV. The objective of this study was to determine whether these cells are protective, utilizing innovative single cell sequencing analyses to probe their functional characteristics.

Methods:
Mononuclear cells from peripheral blood of heart and kidney transplant recipients pre- and three months and one year post-transplant were stimulated with immunodominant CMV peptides. Single IFN gamma+ CMV-responsive T cells were index sorted into 96 well plates, and nested PCR, barcoding, and sequencing performed to analyze T cell receptor (TCR) and a panel of functional genes.

Results:
Expanded CMV-responsive T cells tend to express a limited number of dominant CMV-responsive TCR clones. Clonally expanded CMV-responsive T cells express multiple antiviral proteins. The TCR repertoire may change from pre- to post-transplant. Once established at three months, clonal dominance is maintained until at least one year. The number of antiviral proteins expressed per clonally expanded T cell increases between these time points.

Conclusions:
Protection from CMV post-transplant correlates with maintenance of TCR clonality and increased number of functions per cell. The stability of clonality post-transplant contrasts with the expansion of the total T cell population, and suggests that clonal expansion in the absence of disease recapitulates the pre-existing repertoire. The increased number of functions suggests that the expanded population may become more protective. The potential impact on clinical practice would be to provide further information on the relationship between the T cell response to CMV and long-term outcomes after transplantation.
Abstract 456: Fundamental New Clinical Insight From Excitation-contraction Coupling Studies of the Cardiovascular System via Integrated ECG And Photoplethysmography (PPG) Analyses During Sleep
Authors: Wendelken Daniel R , Sanagala Neha , Conway Ginger A , Jones Steven R , DeMazumder Deeptankar
Published: October 2019

Introduction:
The surface ECG provides critical insights into cardiac excitation. The ECG waveform dynamics inform clinical decisions beyond heart rate and QT measures. Likewise, the PPG waveforms contain physiologically rich information about a person’s health, but it’s clinical use is limited to peripheral oxygen saturation. Importantly, the complexity of ECG and PPG dynamics reflect multi-directional interconnected networks of cellular signaling pathways, tissues, organs and organ system interactions that regulate homeostatic function under changing conditions. While integrated dynamic analysis of ECG and PPG is rooted in fundamental principles of electromechanical coupling and systems biology, this has been unexplored for identifying subclinical illness.

Hypothesis:
Integrated dynamic analysis of ECG and PPG waveforms during sleep identify subclinical cardiovascular illness in a multicenter study of asymptomatic community subjects.

Methods & Results:
We studied polysomnography recordings from 137 adults in a substudy of the multi-ethnic study of atherosclerosis (MESA-5). The cohort had mean age of 69±10 years, 43% women, 11% African Americans, 48% diabetes mellitus (DM) of which 33% received insulin therapy (DM-RxI), 21% prior myocardial infarction (MI), and 16% ostensibly healthy (i.e., unremarkable findings on history, physical exam, lab, etc). Dynamic analysis of PPG waveforms were performed for each sleep stage: (1) Awake before onset of sleep; (2) Non-REM sleep; (3) REM sleep; (4) Arousals during sleep; (5) Awake after completing sleep. In a multivariate model, the odds ratio (5th compared to 1st quintile) for MI for sleep stage 1 was 7.5 (2.1-27), for DM sleep stage 5 was 0.057 (0.0040-0.82), and for DM-RxI for sleep stage 5 was 0.23 (0.057-0.91), with corresponding ROC curve areas ranging between 0.65-0.90 (p < 0.05).

Conclusions:
Integrated dynamic analysis of ECG and PPG waveforms provide fundamental new insight for identifying an individual’s health status, including MI, DM and treatment for DM. This novel strategy has potential for broad clinical application and warrants prospective validation.
Abstract 457: The Effect of Dietary Patterns on Cardiometabolic Risks in Healthy Asian Adults
Authors: Yang Chiu-fen , Lin Tsung-Jen , Liu Chin-Hung
Published: October 2019

Rationale:
Atherosclerosis often leads to acute myocardial infarction and stroke. Diet may be involved in innate immunity-mediated inflammation and the pathogenesis of atherosclerosis.

Objective:
To investigate the effects of various dietary patterns on metabolic profiles, inflammation and atherosclerosis.

Methods and Results:
We began by understanding the dietary pattern using questionnaire for 75 healthy adults who were enrolled in this study. To obtain the metabolic and inflammation profiles, we collected peripheral blood for analysis. The healthiness of vessels was evaluated by carotid intimal-media thickness (CIMT). We found that participants who adhere to vegetarian diet more than ten years showed lower levels of serum total cholesterol compared to omnivores. However, we did not find any beneficial effect of vegetarian diet on other parameters as C-reactive protein, interleukins and CIMT were no difference between two groups. Among “junk foods”, the amount of instant noodles and sugar drink consumption was positively associated with the levels of triglyceride and uric acid. Notably, the increment of sugar drink usage was correlated with higher expression of toll-like receptor (TLR) 2 and TLR4 on peripheral monocytes, but showed no impact on fasting serum glucose. As for the concern of “healthy foods”, participants who took more fresh fruit had significantly better fasting glucose and higher high-density lipoprotein, as well as lower monocyte TLR2 and TLR4 expression. The amount of green vegetable consumption was inversely correlated with low-density lipoprotein and CIMT. In addition to food, water was also important. Participants who drank water more than 2000 ml per day had significant lower CIMT. On the other hand, we did not find any significant effects on coffee consumption. Moreover, for factors that may promote or prevent atherosclerosis, we found that trimethylamine N-oxide and S-adenosyl-L-homocysteine was positively correlated with CIMT whereas L-Lysine and L-Carnitine showed inverse association with CIMT.

Conclusions:
We discovered that consumption of healthy foods and water, but not vegetarian diets per se, is positively correlated with cardiovascular health from the perspective of metabolic panel and inflammation.
Abstract 462: High Speed Imaging of Single Cardiomyocyte Action Potentials Using a Far-red Genetically Encoded Voltage Sensor
Authors: Das Shoshana , Shroff Sanaya , Tseng Hua-an , Chopra Anant , Han Xue , Chen Christopher
Published: October 2019

The ability to image cardiac membrane potentials allows for the observation of cellular communication and electrical activity, both of which are important to maintain cardiac syncytium; these can be altered in diseases (e.g. Long QT Syndrome). Traditionally voltage dyes such as Di-8-ANEPPS have been used to optically measure action potentials (APs). However, these dyes express transiently, have poor signal to noise ratios, and are toxic. More recently, genetically encoded voltage indicators (GEVIs) have been developed to replace state-of-the-art voltage dyes, but sensors currently used within the cardiac field exhibit poor kinetics and/or low signal to noise ratios (SNR). Recently, Archon1, a new genetically encoded voltage sensor, was developed in the neuroscience field; this sensor exhibits excellent membrane localization, temporal sensitivity, and SNR, enabling the optical detection of individual spikes in neurons. Here we use Archon1 for the first time in cardiac cells, to monitor single cell cardiac APs in 2D and 3D in vitro systems in response to different environmental stimuli. Human induced pluripotent stem cell-derived cardiomyocytes were infected with Archon1 and imaged using a one-photon fluorescence microscope equipped with a high speed sCMOS camera to demonstrate cardiac AP tracings. The kinetics and SNR of Archon1 are compared to traditional electrophysiology and Di-8-ANEPPS measurements. Additionally, E-4031 (K+ Channel Blocker) and Nifedipine (Ca2+ Channel Blocker) were used to demonstrate the sensitivity of this sensor in a drug dosage study. To study the APs of single cells within a 3D engineered microtissue, cardiomyocytes expressing Archon1 were seeded into a force transducing micro-pillar device and the APs for optically isolated cells were recorded. Demonstration of this new genetically encoded voltage sensor in cardiac cells enables the monitoring of single and multi-cell APs in 2D and 3D applications and can be extended to in vivo. This tool, newly applied to cardiac biology and tissue engineering will allow for better and more accurate observation of cardiac electrical activity to probe human cardiovascular disease.
Abstract 465: High Circulating Levels of 8-OHdG are an Independent Predictor of Subsequent MACE
Authors: Galenko Oxana , Kergaye Serene A , Knight Stacey , Muhlestein Joseph B , Anderson Jeffrey L , Knowlton Kirk U , Carlquist John F
Published: October 2019

Objective:
Oxidative stress is associated with several cardiovascular risk factors and cardiovascular disease. Damaged cells release cell-free DNA (cfDNA) into the circulation; oxidized cfDNA presence in the plasma indicates a pathological process in the context of oxidative stress. We hypothesized that biomarker of oxidative DNA damage 8-hydroxy- 2’deoxyguanosine (8-OHdG) may be associated with incident major adverse cardiovascular events (MACE).

Methods:
Plasma samples from 84 consenting patients undergoing coronary angiography at Intermountain Healthcare were obtained at the time of the procedure and tested for 8-OHdG by ELISA. Study endpoints were subsequent myocardial infarction (MI), stroke and all-cause death (determined by electronic medical records and death certificates) within median follow-up of 10 years (IQR: 8.8, 10.6). Cox-proportional hazard analysis was used for examining MACE rate association by quartile of 8-OHdG.

Results:
Mean 8-OHdG was significantly higher for individuals with a subsequent MACE vs those without (124.8 and 98.7 nM, respectively; p<0.006, t-test). No other baseline demographic was significantly associated with 8-OHdG. MACE rates by 8-OHdG quartiles revealed a significant trend (p = 0.0346) (Figure). Hazard ratio increased by 1.653 for every 42 unit increase in 8-OHdG, p=0.006.

Conclusion:
High 8-OHdG plasma levels independently predict subsequent MACE. Whether 8-OHdG is contributes etiologically to event causation needs to be determined.
Abstract 472: Smyd1 Variants Regulate Distinct Areas of Chromatin in the Cardiomyocyte
Authors: Szulik Marta , Miller Mickey , Conley Christopher , Parnell Timothy , Franklin Sarah
Published: October 2019

Heart failure is one of the most devastating conditions that contributes to 25% of all deaths in the USA every year. The development of heart failure, which is often preceded by cardiac hypertrophy resulting from an increased workload, is accompanied by global changes in gene expression that resemble a more fetal-like transcriptome. Although many epigenetic factors have been identified, which influence these underlying changes in transcription during disease, we still know very little about how this process is regulated on a molecular level. Smyd1 is a unique histone methyltransferase that modifies lysine residues to regulate gene expression in the cardiomyocyte. Originally, Smyd1 was identified as a necessary regulator of cardiac development, but recently we have shown that Smyd1 is differentially expressed during cardiac hypertrophy and failure and that loss of Smyd1 (in conditional, cardiomyocyte-specific knockout mice) leads to hypertrophic growth, metabolic dysfunction and ultimately heart failure. In mice, the
smyd1
gene produces two variants, Smyd1a and Smyd1b, which differ only by a 13 amino acid deletion. In addition, we have shown that overexpression of Smyd1a (but not Smyd1b) can prevent phenylephrine-induced hypertrophy, however, both variants are capable of suppressing ANF expression. Despite this interesting data, the specific genomic regions bound by these variants and the unique genes they regulate are unknown. Therefore, to identify the genes regulated by Smyd1 variants we performed ChIP-Seq in cardiomyocytes under normal and hypertrophic conditions. Interestingly, our analyses show that Smyd1a binds DNA more frequently than Smyd1b under basal conditions (2,392 vs 1,004 binding events, respectively) but that Smyd1b exhibits greater binding in hypertrophic conditions (2,414 vs 5,045 binding events). Moreover, bioinformatics analysis of these sites confirmed both conserved and unique binding preferences and showed that while both variants are most abundant in distal intergenic regions (32-57%), Smyd1a is uniquely enriched (24%) at gene promoters (<1kb). These exciting results suggest novel and distinct regulatory functions of Smyd1 isoforms and begin to inform us how Smyd1 regulates cardiac morphology and physiology.
Abstract 479: Cardiomyopathy Phenotypes Observed in Human Engineered Heart Tissue Depend on Functional Maturation
Authors: Sewanan Lorenzo R , Shen Shi , Ng Ronald , Li Xia , Campbell Stuart G
Published: October 2019

Human iPSC-based models of cardiomyopathies have become increasingly prevalent. These models enable the study of genotype-phenotype relationships within a human context and have already led to new insights. A potential limitation of iPSC-derived cardiomyocytes (iPSC-CMs) is their varying degrees of functional maturation with respect to electrophysiology, calcium handling, and contractility. Variable levels of maturity may explain how different groups investigating hypertrophic cardiomyopathy (HCM) mutations in iPSC cardiomyocytes have arrived at conflicting results. Nevertheless, whether or not advanced functional maturation truly impacts disease phenotype in iPSC cardiomyocyte models remains an open question. Using a novel protocol that combines pacing and physiological media in a three-dimensional engineered heart tissue (EHT) derived from decellularized myocardium, we are able to mature human engineered heart tissues within three weeks to recapitulate key functional characteristics of adult heart tissue, such as a robustly positive force-frequency response, a strong post-rest potentiation, potent calcium handling and excitation-contraction handling, and mature isometric twitches that closely match that of intact adult human ventricular myocardium. Using our platform, we investigated the effect of maturation on the functional consequence of a classic variant (E62Q) in the thin filament protein alpha-tropomyosin (TPM1). Initial characterization of TPM1 E62Q in EHTs show a hypercontractile phenotype compared to its isogenic wild-type line, with limited changes in calcium handling. Preliminary data on maturing TPM1 E62Q EHTs show that additional hallmarks of disease may develop with maturation such as arrhythmogenicity, hypertrophy, and fibrosis. In our work, we develop a method for advanced maturation of hiPSC-CMs in EHTs and show the dependence on maturation status of progressive hallmarks of HCM in human EHTs compared to EHTs grown under conditions standard for the field.
Abstract 480: A Novel Role for Telomerase in Calcific Aortic Valve Disease
Authors: Hortells Luis , Boufford Camille , Regan Cailyn , Chu Claire , Moorhead William J , Doyon Genevieve , Bruemmer Dennis , St Hilaire Cynthia
Published: October 2019

Intro:
Telomerase (TERT) is an enzyme best known for its telomere-extending activities on the ends of chromosomes, however, less known are the non-canonical, transcriptional and epigenetic actitivies of TERT. We sought to assess if TERT, through non-canonical activities, contributes to the progression of calcific aortic valve disease (CAVD). It has been established that overexpression of TERT in mesenchymal stem cells primes these cells to differentiate into osteoblasts. Other studies have identified non-canonical roles for TERT in inducing the transcription of genes in inflammatory and cell differentiation pathways.

Methods:
Using mesenchymal stem cells, primary valve interstitial cells (VICs) from control and CAVD patients, and smooth muscle cells (SMCs) from WT and TERT knockout mice, we performed in vitro biochemical assays to study the role of TERT in the calcification process.

Results:
Our data shows that TERT is highly expressed in CAVD valves compared to Control valves. Under osteogenic differentiation conditions Control valve interstitial cells (VICs) upregulate TERT protein levels and calcify. Inflammatory signals induce TERT expression in VICs and exacerbates calcification. Similarly, WT vascular VSMCs readily calcify in vitro, but VSMCs from TERT knockout mice do not, and TERT deletion also reduced valve calcification in LDLR/TERT double knock mice. These studies provide evidence that TERT is necessary is the osteogenic switch of a healthy to a calcifying VIC. Knocking down TERT reduces expression of the osteogenic transcription factor RUNX2 and we further provided evidence that STAT5 may help to mediate TERTs effects on osteogenic gene transcription.

Conclusion:
From these data we suggest that TERT is required for valve calcification by inducing the osteogenic transition of quiescent valve interstitial cells (VICs) into calcifying VICs, and that TERT and STAT5 co-regulate transcription of osteogenic genes. These results indicate that TERT is an active contributor to the calcification process of valve tissues and our futures studies will delineate the mechanisms involved.
Abstract 481: Investigation into the Genetic Cause of Congenital Dilated Cardiomyopathy Using Human Induced Pluripotent Stem Cells
Authors: Wang Kuo-chen , Chun Young Wook , Hong Charles C
Published: October 2019

Congenital dilated cardiomyopathy (cDCM) is a rare but typically fatal disease. In most cases, its etiology is unknown, but a genetic root cause is often suspected. The objective of current study is to determine whether a novel non-structural gene causes cDCM. We hypothesized that rare genetic mutations caused cDCM, which could be modeled using patient-derived induced pluripotent stem cells (iPSCs). We generated the cardiomyocytes from iPSCs of a cDCM proband and found significant impairment in contractility and mitochondrial function, compared to those of healthy controls. To identify the causal mutations, we performed a whole exome sequencing of the cDCM patient and his parents (“trio”). Based on the assumption of recessive mode of inheritance or a
de novo
mutation, 9 candidate causal genes were identified. On the ground of the expression profiles of trio and pathogenicity predictor algorithms, we identified an indel and a nonsynonymous point mutation in the rotatin (encoded by
RTTN
) gene as the putative genetic defect responsible for cDCM. CRISPR/Cas9-mediated knockout of
RTTN
in healthy control iPSCs recapitulated cardiomyocyte defects, and the correction of the missense mutation in the disease iPSCs restored cardiomyocyte structure and function, confirming causality. Thus, rotatin is a new causal molecule for cDCM and plays an important role in cardiac regulation.
Abstract 485: STING(ing) the Heart: How DNA Damage and Inflammation Orchestrate Cardiac Remodeling and Heart Failure
Authors: Al-Khalaf Mohammad H , Akolkar Gauri , Liu Peter P
Published: October 2019

Background:
We aimed to investigate the role of DNA breaks in inducing inflammation leading to adverse remodeling, premature senescence, and cardiac dysfunction leading to onset of heart failure.

Methods:
in vivo
: Heart-focused IR (using XRad225Cx from PXi) on adult Sprague Dawley rats; 4-5 per group; dose: 0, 15, 30, 45 Gy (endpoint 12 weeks post-IR). Serial echocardiogram and blood collection, P/V at endpoint; whole heart harvest analysis.
in vitro
: Primary cardiomyocytes from adult rats; human derived cardiac AC16 cell line; UVB ionizing radiation; protein extraction, western probing; gene expression analysis; immunofluorescence staining and microscopy.

Results:
DNA damage response, as illustrated by XRCC1 protein expression, is upregulated in multiple heart injury/failure models (TAC, MI, aging) and differ depending on cell stage and/or age (
in vitro
proliferative vs differentiated AC16 cells). Using
in vitro
UVB radiation, exposure alters cardiomyocyte morphology, illustrated by α-actinin immunostaining, and activates innate inflammation and senescence, as illustrated by westerns and immunostaining of cGAS, STING, p16 and other relevant proteins. We demonstrate using heart-focused ionizing radiation a novel technique to induce aging related DNA damage and onset of diastolic heart dysfunction. Serial ECG show significant increase in E/A ratios at 8- and 12-week post irradiation of 30 and 45 Gy (E/A>2.0). We show P/V analysis supporting ECG data (~50% decrease in dPdt min, 2-fold increase in Tau, and significant [p<0.01] increase in End diastolic pressure, and no change in End systolic pressure). Using an inhibitor targeting cytosolic DNA sensor cGAS, we show attenuation of cardiac remodeling and expression of senescence.

Conclusions:
We elucidate the molecular pathway originating from the onset of DNA damage accumulation, leading to activation of specialized innate immunity proteins that ultimately upregulate inflammation and senescence. Here we uncover and connect key interactions between multiple pathways converging towards heart failure, and provide evidence for a novel therapeutic drug targeting cardiac aging.
Abstract 491: Small Gtpase Rhoe Regulates Inflammatory Response in Myocardial Infarction
Authors: Luo Weijia , Dai Yuan , Song Jiangping , Chang Jiang
Published: October 2019

Background:
Myocardial infarction (MI) is a severe clinical condition caused by coronary artery thrombotic occlusion. An acute inflammatory response to MI is essential for cardiac healing, while excessive and prolonged inflammation causes cytotoxic damages and provokes adverse cardiac remodeling. In the present study, we demonstrate a novel inflammatory mediator, RhoE, and its cardioprotective role in MI.

Methods:
We use three genetic mouse lines in this study: global RhoE knockout, cardiac-specific RhoE haploinsufficient, and cardiac-specific RhoE overexpression mice. Cardiac function and inflammation in the first week post MI are assessed in mice receiving left anterior descending artery (LAD) ligation. Mechanistic study is conducted through a set of molecular signaling experiments including bimolecular fluorescence complementation (BiFC), immunoprecipitation, electrophoretic mobility shift assay and mRNA microarray analysis. Finally, we investigate the expression and clinical significance of RhoE in MI patients.

Results:
RhoE is upregulated in mouse post MI. RhoE deletion leads to upregulation of pro-inflammatory genes in mouse embryo heart. RhoE haploinsufficiency causes excessive inflammatory response with deleterious cardiac function post MI, while RhoE overexpression restrains post-MI inflammation and preserves cardiac function and survival. Mechanistically, RhoE specifically inhibits NF- κB activation in vivo and in vitro. RhoE binds to p65 and p50 individually in cytosol, blocking their dimerization and nuclear translocation. Consistent with findings in the mouse MI model, MI patients with higher RhoE expression show diminished cardiac inflammation and consequently a better prognosis.

Conclusions:
This study identified RhoE as a fine-tuning factor modulating post-MI inflammation. RhoE expression level in heart positively correlates with the outcomes of MI patients.

Impact:
The discovery of RhoE provides a potential therapeutic target for MI. RhoE abundance in the heart post-MI might be used for prognosis assessment.
Abstract 493: Multidirectional Analysis of Anti-atherosclerotic Effect Caused by Korean Radish in Apo E–/– Mice Model
Authors: Na Jinhyuk , Khan Adnan , Hwang Kyung-A , Park Youngja H
Published: October 2019

Atherosclerosis, a leading cause of worldwide mortality and morbidity, is mainly controlled with statins, however, the statins have severe side effects like rhabdomyolysis. In search of an alternative for statin, we investigated anti-atherosclerotic and anti-hyperlipidemic effects of two different species of
Raphanus sativus Linn
(also known as radish), Jeju (white radish) and Ganghwa (purple radish) cultivated in South Korea. An atherosclerosis model in
ApoE
knockout mice was firstly established with high fat diet, followed by feeding the mice with extracts of each radish. Concentration of plasma lipids and histological changes were evaluated. Metabolomics analysis of urine was determined to see the effect of radish. Ganghwa-radish treated mice were observed with significantly decreased levels of total cholesterol, low-density lipoprotein, triglyceride, atherogenic index and cardiac risk factor while high-density lipoprotein was significantly increased. Steroid biosynthesis and steroid hormone biosynthesis were although significantly altered metabolic pathways among both radish treated groups, however, significant changes in these pathways were mainly observed with the treatment of Ganghwa-radish. Crucially, estrogens, estrone and estradiol, were significantly decreased in Ganghwa-radish treated group compared to control. In conclusion, we confirmed the prominent antilipidemic effect of Ganghwa-radish compared with Jeju-radish and such results might be due to the inhibition of estrogens by Ganghwa-radish treatment.
Abstract 494: Role of Slug / PIP Axis in Pulmonary Hypertension Secondary to Pulmonary Fibrosis
Authors: Ruffenach Gregoire , Vaillancourt Mylene , Hong Jason , Cao Nancy , Cunningham Christine , Saggar Rajan , Reddy Srininivasa , Umar Soban , Fishbein Gregory , Eghbali Mansoureh
Published: October 2019

Pulmonary hypertension (PH) secondary to pulmonary fibrosis (PF-PH) is the second most common cause of PH. While never studied in detail, pathology reports suggest that vascular remodeling (VR) differs between PF and PF-PH. In PF, VR is mainly limited to fibrotic areas, whereas in PF-PH, it also exist in non-fibrotic areas. These histological differences suggest potential molecular differences. To better understand this mechanism, we investigated the expression of the transcription factor Slug which is known to play a role in PH and PF.

Using explanted rat (n=7/group) and human (n=7-14/group) lungs, we compared lung fibrosis, VR and Slug expression between non-fibrotic and fibrotic areas in PF and PF-PH. Online microarray data (GSE24988) were used to find the targets of Slug that are implicated in VR. A new animal model recapitulating our findings in patients was used to test the therapeutic potential of Slug inhibition (n=10/group). This model is based on intra-tracheal instillation of bleomycin (2.5mg/Kg) at day 0 and 2weeks later, an injection of monocrotaline (60mg/kg). PH was assessed by right ventricular systolic pressure (RVSP). P<0.05 are considered significant.

In both PF and PF-PH patients, fibrotic areas (PF29±4; PF-PH37±3) exhibit significantly increased VR when compared to non-fibrotic areas of the lung (PF 22±1; PF-PH 31±6). PF-PH patients have increased pulmonary vascular thickening in both areas vs PF patients. This is concomitant with an increased number of Ki67+ vascular cells in PF-PH (12±2%) vs PF (8±1%) as well as an upregulation of Slug in PF-PH patients (2.3±0.5) vs PF (1±0.1). Co-immunolabeling with CD68 demonstrate that macrophages are the main cell type responsible for Slug up-regulation in PF-PH. Human microarray data reveal an up-regulation of the Prolactin-induced protein (PIP) in PF-PH vs PF (9±3 vs 1±0.4). PIP is an extracellular transcriptional target of Slug, known to promote cell proliferation. In-vitro, PIP significantly increases pulmonary arterial smooth muscle cell proliferation in a dose dependent manner. Finally, Slug inhibition decreases RVSP (47±3 vs 62±3mmHg) in an animal model of PF-PH.

There are histological differences between PF-PH and PF lungs that are at least in part mediated by a Slug/PIP axis leading to VR.
Abstract 496: Inhibition of the Rna Binding Protein Hur Protects Against Cardiac Ischemia/reperfusion Injury by Reducing Inflammatory Gene Expression
Authors: Slone Samuel , Fleifil Salma , Anthony Sarah , Green Lisa , Nieman Michelle L , Lorenz John , Tranter Michael
Published: October 2019

Despite medical advances, cardiac ischemia/reperfusion (I/R) injury remains a leading cause of morbidity and a huge economic burden in the United States. RNA binding proteins are becoming recognized as potential mediators of cardiac physiology and pathology, but the role of HuR, an RNA binding protein highly expressed in myocytes, in acute cardiac I/R injury is unknown. HuR has been shown in other tissues to be a critical post-transcriptional mediator of pro-inflammatory chemokine and cytokine gene expression, and we have shown HuR to be activated (nuclear-to-cytoplasmic translocation) in cardiomyocytes 2 hours post-ischemia/reperfusion injury.

To address the functional role of HuR in I/R, cardiomyocyte-specific HuR deletion mice (iCM-HuR
-/-
) were subjected to 30 minutes of LAD (left anterior descending) coronary artery ligation followed by 24 hours of reperfusion. In parallel, a separate group of wild-type mice were subjected to 30 minutes ischemia with a pharmacological inhibitor of HuR given just prior to reperfusion. Analysis of infarct size showed a smaller infarct with both HuR genetic deletion (~10% decrease in infarct size compared to control, N=3, P<0.05) and pharmacological inhibition (~11% decrease in infarct size compared to vehicle, N=4, P=0.069). Similarly, HuR inhibition significantly reduced cell death, caspase-3 activity, and inflammatory gene expression in an
in vitro
model of simulated I/R using neonatal rat ventricular myocytes. HuR inhibition results in a significant blunting of IL-6 and TNF-alpha gene expression two hours post-reperfusion
in vivo
, suggesting that HuR activity is necessary for the early induction of inflammatory gene expression networks. In addition, it appears that HuR inhibition attenuates macrophage infiltration following ischemia/reperfusion injury. In conclusion, our results suggest that inhibition of HuR is protective against cardiac I/R injury through a reduction in inflammatory gene expression.
Abstract 502: Reversible Cardiac Action Potential Recordings to Track Cellular Electrophysiology Over Days
Authors: Zlochiver Viviana , Edwards Stacie , Cook Jonathan A , Joshi-Mukherjee Rosy
Published: October 2019

Long-term action potential (AP) studies from the same cell site over days in multicellular preparations are currently lacking, thus limiting our understanding of cardiac physiology in development and disease. We recently reported a novel high-throughput platform for label-free AP measurements using multielectrode array (MEA) technology to gain intracellular access by way of electroporation. We hypothesized that multiple reversible electroporations of the same cell site will allow high-resolution recordings of AP waveforms without detrimental effects to the cell membrane. A total of 19 hiPSC-CM constructs on multiwell MEAs were simultaneously electroporated by employing a train of low voltage pulses (1 V, 1 ms, 1 Hz) for 30 s to elicit a transient transformation of field potentials (FPs) to APs. Waveforms were recorded from 12 electrodes per construct for electrophysiological characterization of the network. Recurrent electroporations of the same cell site at 0, 24, 48, 72 and 96 h enabled tracking of the electrophysiological evolution of the AP waveforms (see figure). We analyzed millivolt AP signals 10 s following the end of electroporation before APs reverted to FP signals strongly suggesting cell membrane recovery and network integrity for subsequent recordings. This methodology allows for screening transmembrane APs from cardiomyocyte networks over days. Overall, we present a spatio-temporal model capable of tracking FPs and APs in a non-invasive approach for cardiomyocyte functional maturation, developmental and pharmacological studies.