Olivia T Ly¹, Grace Brown², Hanna Chen³, Liang HONG⁴, Xinge Wang, Yong Duk Han⁴, Mahmud Arif Pavel¹, Arvind Sridhar⁵, Mark Maienschein-Cline³, Sang Ging Ong¹, khaled Abdelhady, Malek G Massad⁶, Lona Ernst Rizkallah¹, Jalees Rehman⁴, SALMAN KHETANI, Dawood Darbar⁴; ¹Univ of Illinois COM, Chicago, Chicago, IL, ²Univ of Illinois at Chicago, Orland Park, IL, ³Chicago, IL, ⁴Univ of Illinois at Chicago, Chicago, IL, ⁵Univ of Illinois, Chicago, Chicago, IL, ⁶Univ of Illinois Chicago, Chicago, IL,

Introduction: Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) are ideally suited for modeling heritable atrial fibrillation (AF) and testing personalized pharmacological therapy, but there is limited data on the maturation of iPSC-atrial (a) CMs. While we reported the only non-ion channel gene (NPPA), encoding atrial natriuretic peptide (ANP), as a cause of familial AF, the underlying cellular mechanisms remain unclear. Immaturity of iPSC-aCMs is one potential explanation. Hypothesis: We hypothesized that a combinatorial engineering approach to maturation with biochemical cues (T3 [T], IGF-1[I], dexamethasone [D]; [TID]), bioenergetic supplement (fatty acids [FA]), and electrical stimulation (ES), synergistically promotes the electrophysiological (EP), structural, and metabolic maturity of iPSC-aCMs to a level comparable to human atrial tissue (HAT) and will unmask the underlying cellular mechanisms of an NPPA mutation. Methods: We differentiated iPSC-aCMs with retinoic acid, applied TID+FA+ES, and compared the EP (patch clamping, optical voltage mapping), structural (immunofluorescence, western blots [WB]), metabolic (Seahorse Analyzer, WB, RT-PCR), and transcriptomic (RNA-seq) maturity with immature iPSC-aCMs and adult human aCMs from the same patient. We generated iPSC-aCMs from a family carrying the NPPA-S64R mutation, an isogenic control using CRISPR-Cas9, and used our matured iPSC-aCMs to elucidate the EP signature of AF. Results: TID+FA+ES treated iPSC-aCMs displayed improved sarcomeric organization and oxidative capacity (Figure 1a,b), more hyperpolarized RMP and increased APA (Figure 1c,d). Only mature NPPA-S64R iPSC-aCMs prolonged APD, and increased I_Ks density when compared to immature aCMs (Figure 1e,f,g,h). Conclusion: We established a combinatorial engineering approach that promoted the comprehensive maturation of iPSC-aCMs and unmasked the underlying mechanism of an AF-causing NPPA mutation.

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Key Words: Atrial fibrillation; Cellular Electrophysiology; Stem cell biology; Gene mutations; Cellular Engineering

Camila Hartmann¹, Anna Flavia Ribeiro dos Santos Miggiolaro¹, Jarbas da Silva Motta Junior², Lucas Baena Carstens³, Caroline Busatta Vaz De Paula³, Sarah Fagundes Grobe¹, Larissa Hermann de Souza Nunes², Gustavo Lenci Marques¹, Peter Libby⁴, Lídia Zytynski Moura¹, Lucia de Noronha³, Cristina Pellegrino Baena¹; ¹PUCPR; Marcelino Champagnat Hosp, Curitiba, Brazil, ²Marcelino Champagnat Hosp, Curitiba, Brazil, ³PUCPR, Curitiba, Brazil, ⁴Brigham and Womens Hosp, Cambridge, MA

Introduction: Myocardial injury associates significantly and independently with mortality in COVID-19 patients. However, the pathogenesis of myocardial injury in COVID-19 remains unclear, and cardiac involvement by SARS-CoV-2 presents a major challenge worldwide. Hypothesis: This histological and immunohistochemical study sought to clarify the pathogenesis and propose a mechanism with pathways involved in COVID-19 myocardial injury. Methods: Postmortem minimally invasive autopsies were performed in six patients who died from COVID-19, and the myocardium samples were compared to a control group (n=11). Histological analysis was performed using hematoxylin-eosin and toluidine blue staining. Immunohistochemical (IHC) staining was performed using monoclonal antibodies against targets: caspase-1, caspase-9, gasdermin-d, ICAM-1, IL-1β, IL-4, IL-6, CD163, TNF-α, TGF-β, MMP-9, type 1 and type 3 collagen. The samples were also assessed for apoptotic cells by TUNEL. Results: Histological analysis showed severe pericardiocyte interstitial edema and higher mast cells counts in all COVID-19 myocardium samples. The IHC analysis showed increased expression of caspase-1, ICAM-1, IL-1β, IL-6, MMP-9, TNF-α, and other markers in the hearts of COVID-19 patients. Expression of caspase-9 did not differ from the controls, while gasdermin-d expression was less. The TUNEL assay was positive in all the COVID-19 samples supporting endothelial apoptosis. Conclusions: The pathogenesis of COVID-19 myocardial injury does not seem to relate to primary myocardiocyte involvement but to local inflammation with associated interstitial edema. We found heightened TGF-β and interstitial collagen expression in COVID-affected hearts, a potential harbinger of chronic myocardial fibrosis. These results suggest a need for continued clinical surveillance of patients for myocardial dysfunction and arrythmias after recovery from the acute phase of COVID-19.

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Key Words: Myocardium; COVID-19; Endothelium; Fibrosis; Pathology

Seulhee Kim¹, Patrick Ernst², Yingnan Si¹, Taehyun Kim¹, Min Xie¹, Margaret Liu², Lufang Zhou¹; ¹Univ of Alabama Birmingham, Birmingham, AL, ²Univ of Alabama at Birmingham, Birmingham, AL,

Background: While mounting evidence indicates that mitochondria, the cell powerhouse, is involved preconditioning-mediated cytoprotection, the role of mitochondrial membrane potential (ΔΨ_m) in this process is not well defined. Recently, our lab developed a novel mitochondrial-targeted optogenetic technology (mOpto) that can induce light intensity-dependent dynamic ΔΨ_m depolarization in a variety of cell types. The aim of this study was to utilize mOpto to examine the role of ΔΨ_m preconditioning in protecting cells against stress-induced injury. Methods: Cardiomyocytes expressing mOpto gene were subjected to transient, low intensity LED illumination (3 hours, 0.2 mW/mm²), followed by exposing to various stress such as FCCP (20 μM, 24 hours), H₂O₂ (200 μM, 24 hours), or ischemia-reperfusion (I2/R4: glucose-free medium and 0% O₂ for 2 hours, and complete culture medium and oxygenated for 4 hours). Cell viability, ΔΨ_m and reactive oxygen species (ROS) were measured before and after stress. Results: Our data showed that cell viability is significantly higher in preconditioned cells compared to the control groups post I2/R4 treatment, along with reduced ROS level and alleviated ΔΨ_m depolarization (see figure). The mOpto-mediated preconditioning also protected cells from FCCP and H₂O₂ treatments to different extents. Analysis performed immediately after preconditioning indicated a slight ROS increase and ΔΨ_m dissipation, compared to controls. Moreover, Parkin-deficiency attenuated mOpto preconditioning-mediated cytoprotection, suggesting involvement of Parkin-dependent signaling cascades such as mitophagy. Conclusion: mOpto-medicated ΔΨ_m preconditioning effectively protected cardiomyocytes from cell injury induced by various stress, likely through Parkin-dependent mitophagy. Future studies will determine whether this process is ROS-dependent.

This research has received full or partial funding support from the American Heart Association.

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Key Words: Mitochondria; Preconditioning; Cell physiology

Ravi K Adapala¹, Venkatesh Katari¹, Vahagn A Ohanyan², William M Chilian³, Sailaja M Paruchuri⁴, Charles Thodeti¹; ¹Univ of Toledo, Toledo, OH, ²Northeast Ohio Med Univ, Rootstown, OH, ³NORTHEAST OHIO MEDICAL UNIV, Rootstown, OH, ⁴UNIVERSITY OF TOLEDO, TOLEDO, OH,

Prolonged changes in hemodynamic pressure due to pathological insults disrupts the electrical and biomechanical coupling between cardiomyocytes which leads to heart failure. However, the mechanotransduction pathways that regulate cardiomyocyte hypertrophy in response to pathological stress are still unknown. Transient receptor potential vanilloid 4 ion channel (TRPV4) is a mechanosensitive ion channel implicated in cardiac fibrosis and cardiomyocyte dysfunction. We have recently shown that global deletion of TRPV4 preserves cardiac function and structure via reducing cardiac fibrosis after MI. Here, we investigated cardiomyocyte specific role of TRPV4 following pressure overload (transverse aortic constriction; TAC) induced hypertrophy. TRPV4 expression was increased in the hearts of wild type (WT) mice following TAC with concomitant increase in myocyte cross-sectional area, cardiac fibrosis and reduced ejection fraction. However, myocardial structure and function was preserved in global TRPV4KO mice compared to WT, 28 days after TAC. To investigate cardiomyocyte specific role of TRPV4, we have generated cardiomyocyte specific TRPV4 knockout mice (TRPV4^CMKO) by crossing TRPV4^lox/lox with Myh6^cre mice. First, TRPV4 deletion in cardiomyocytes was confirmed through genotyping and real time PCR in tail snips and isolated cardiomyocytes. Next, we subjected the TRPV4^lox/lox and TRPV4^CMKO to TAC and then measured cardiac function. We found that cardiac hypertrophy (cardiomyocyte cross sectional area) and cardiac fibrosis was increased in both TRPV4^lox/lox and TRPV4^CMKO mice compared to respective sham controls. In contrast, absence of TRPV4 in cardiomyocytes preserved the cardiac function compared to TRPV4^lox/lox mice. Further RNA sequencing analysis revealed that TAC upregulated hypertrophic genes in TRPV4^lox/lox hearts but downregulated in TRPV4^CMKO hearts. Our results indicate that cardiomyocyte specific deletion of TRPV4 alone preserves cardiac function despite of increased remodeling of the heart by downregulating hypertrophic genes and identify TRPV4 as new cardiomyocyte therapeutic target for the heart failure.

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Key Words: Calcium; Cardioprotection; Cardiac hypertrophy; Fibrosis

Qutuba Karwi¹, Keshav Gopal¹, Seyed Amirhossein Tabatabaei Dakhili¹, Cory S Wagg², Riccardo Perfetti³, Ravichandran Ramasamy⁴, John R Ussher¹, Gary D Lopaschuk¹; ¹Univ of Alberta, Edmonton, Canada, ²Univ of Alberta, Edmonton Ab, Canada, ³Applied Therapeutics, New York, NY, ⁴NYU Grossman Med Cntr, New York, NY,

Introduction: Diabetic cardiomyopathy (DbCM) increases mortality and morbidity in type 2 diabetes (T2D) subjects. Increased aldose reductase (AR) activity has been linked to the impairment of cardiac energy metabolism and function in DbCM subjects. AR inhibition has been shown to be cardioprotective in DbCM mice. Dapagliflozin, a sodium-glucose cotransporter inhibitor, reduces the risk of cardiovascular death independent of diabetes status. Hypothesis: Concomitant administration of the AR inhibitor (AT-001) with dapagliflozin improves cardiac function and decreases diastolic dysfunction in DbCM. Methods: DbCM was established in human AR overexpressing transgenic (hAR-Tg) mice by subjecting them to a high-fat diet (60% kcal from lard) for 10-wk with a single intraperitoneal streptozotocin injection (75 mg/kg) at 4-wk. Male mice (n=4-6/group) were randomized to receive either vehicle, AT-001 (40 mg/kg/day), dapagliflozin (1 mg/kg/day), or a combination of AT-001plus dapagliflozin for 3-wk. Results: AT-001 treatment decreased cardiac fatty acid oxidation in DbCM mice, an effect that was associated with a reduction in pathological cardiac remodelling as indicated by a change in left ventricular mass. Cardiac efficiency (cardiac work/TCA activity) was also improved by AT-001 treatment. These benefits were unique to the treatment with AT-001. When potential synergistic effects were examined, cardiac function was significantly improved in the AT001+dapagliflozin-treated DbCM mice, as reflected by an improved diastolic function in vivo (a decrease in the E/e’ ratio) and an increase in cardiac work in isolated working hearts (2.4 ± 0.04 vs 1.9 ± 0.03 joules/min/g dry wt) compared to the dapagliflozin-treated DbCM mice, p<0.05). This cardioprotection did not involve changes in myocardial oxygen consumption in the DbCM mice and treatment with AT001+dapagliflozin significantly enhanced cardiac efficiency (cardiac work/O₂ consumption) in the DbCM mice (61.54 ± 0.7 vs 52.63 ± 2.0 μmol/min/g dry wt in the dapagliflozin-treated DbCM mice, p<0.05). Conclusion: AT-001 provides a unique profile in halting the progression of DbCM. AT-001 both alone and in combination with SGLT-2 inhibition may be beneficial in targeting the pathogenesis of DbCM.

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Key Words: Cardiac metabolism; Cardiomyopathy; Diabetes (Type II); Sodium glucose co-transporter 2 inhibitors

Seung-Jun Lee¹, Subin Jung¹, Tae Wook Noh¹, Hyo-Hyun Kim¹, Jung-Hwan Kim¹, Hyun-Chel Joo¹, Chul-min Ahn², Young-Guk Ko³, Donghoon Choi⁴; ¹Severance Cardiovascular Hosp, Seoul, Korea, Republic of, ²Yonsei Univ Health System, Seoul, Korea, Republic of, ³Yonsei Cardiovascular Cntr, Seoul, ⁴Severance Hosp, Seoul, Korea, Republic of

Background: Sacubitril/Valsartan (Sac/Val), an angiotensin II (Ang II) receptor-neprilysin inhibitor, is a potent drug for heart failure. However, its potential benefit in other cardiovascular disease is not understood. We sought to investigate the pathogenetic role of neprilysin in abdominal aortic aneurysm (AAA), and therapeutic effect of Sac/Val for amelioration of AAA progression. Methods and Results: Expression of neprilysin was investigated using human AAA tissue undergoing graft replacement and normal human aorta acquired during heart transplantation. Immunoblotting (Figure 1A) and immunofluorescence staining demonstrated a strong expression of neprilysin in AAA tissues, particularly in the vascular smooth muscle cell (VSMC). Sixteen weeks old ApoE^-/- male mice fed an atherogenic diet were administered with vehicle, Val (26 mg/kg/day), or Sac/Val (31 mg/kg/day of Sac, 26 mg/kg/day of Val) for 28 days with Ang II infusion (1000 ng/kg/min). Assessment for the progression of AAA via in vivo magnetic resonance imaging and ex vivo histological analysis (Figure 1B) revealed that the maximal diameter of AAA was markedly suppressed by Sac/Val treatment compared with Val (0.87±0.05 vs. 1.28±0.16 mm, p=0.018, n=8 for each group), or vehicle (0.87±0.05 vs. 1.52±0.16 mm, p<0.001, n=8). Mechanistically, neprilysin upregulation in human aorta promoted the apoptosis of VSMC (Figure 1C), an integral component of vascular integrity, via PDGF-B dependent mechanism. Gene-set enrichment analysis also demonstrated upregulation of genes related to apoptosis of VSMC in the human VSMC transfected with neprilysin. Conclusion: We demonstrate a distinct expression of neprilysin in human AAA tissue, and the pharmacological inhibition of neprilysin substantially alleviated the progression of AAA in murine AAA model, thus paving a way for future application of Sac/Val for treatment of AAA to avoid high-risk vascular surgery.

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Key Words: Abdominal aortic aneurysm; Vascular medicine

Virginie Montiel¹, Irina Lobysheva², Ludovic Gérard³, Marjorie Vermeersch⁴, David Perez-Morga⁴, Thomas Castelein³, Jean Baptiste Mesland³, Philippe Hantson³, Christine Collienne³, Damien Gruson³, Marie-Astrid van Dievoet³, Alexandre Persu⁵, Christophe Beauloye⁶, Mélanie Dechamps⁷, Leïla Belkhir³, Annie Robert⁸, Marc Derive⁹, Pierre-François Laterre³, A.H.J Danser¹⁰, Xavier Wittebole³, Jean-Luc Balligand¹; ¹FATH - IREC / Cliniques Universitaires Saint Luc, Brussels, Belgium, ²FATH - IREC, Brussels, Belgium, ³Cliniques Universitaires Saint Luc, Brussels, Belgium, ⁴ULB, Gosselies, Belgium, ⁵Cliniques Universitaires Saint-Luc, Brussels, Belgium, ⁶IREC - UCL, Brussels, Belgium, ⁷CARD - IREC / Cliniques Universitaires Saint Luc, Brussels, Belgium, ⁸EPID - IREC, Brussels, Belgium, ⁹Inotrem SA, Vandoeuvre-les-Nancy, France, ¹⁰Erasmus MC, Rotterdam, Netherlands

Introduction: SARS-CoV-2 targets endothelial cells through the angiotensin-converting enzyme 2 receptor. Hypothesis: The impact of the resulting endothelial injury is currently unknown but may contribute to the pro-coagulant state classically described during Covid-19. Methods: We prospectively compared clinical and biological parameters in 30 ICU- and 30 non-ICU-admitted Covid-19 patients, 10 ICU-admitted patients with septic shock unrelated to Covid-19 and 15 matched control subjects for similar cardiovascular risk factors as ICU Covid-19. Main Results: Early SARS-CoV-2 infection was associated with an imbalance between an exacerbated oxidative stress (plasma peroxides levels in ICU patients vs. controls: 1456.0 ± 400.2 vs 436 ± 272.1 mmol/L; P< 0.05) and a reduced nitric oxide bioavailability (nitrosylated hemoglobin, HbNO) proportional to disease severity (HbNO in ICU patients vs. controls: 116.1 ± 62.1 vs. 163.3 ± 46.7 nmol/L; P< 0.05). HbNO levels inversely correlated with oxygenation parameters in Covid-19 patients. Plasma levels of angiotensin II (ICU patients vs. controls: 1.2 ± 1.0 vs. 2.0 ± 1.1 fmol/m; P< 0.05), aldosterone (235.1 ± 145.1 vs. 284.4 ± 181.4 pg/mL; P=NS), renin (20.0 ± 22.4 vs 19.7 ± 13.2 pg/mL; P=NS) or serum level of TREM-1 (ICU patients vs. ICU septic shock unrelated to Covid-19: 265.3 ± 161.2 vs 608 ± 338.3 pg/mL; P< 0.05) ruled out any hyper-activation of the renin-angiotensin-aldosterone system or the leucocyte respiratory burst in ICU Covid-19 patients, contrary to septic patients. Electron microscopy illustrated irregular aspect of the endothelial wall due to fibrillar network of fibrin depots and damaged but viable endothelial cells responsive to circulating autacoids. Conclusion: Endothelial oxidative stress with ensuing decreased NO bioavailability appears as a key pathogenic factor of endothelial dysfunction and respiratory failure in ICU Covid-19 patients that, contrary to septic shock is not associated with an overactivation of the renin-angiotensin-aldosterone system or leucocyte respiratory burst. These results highlight an urgent need for oriented research leading to a better understanding of the specific endothelial oxidative stress that occurs during SARS-CoV-2.

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Key Words: Endothelial function; COVID-19; Oxidative stress; Thrombosis; Angiotensin II

Bin Liu, Shuai Li, Dan Yi, Jiakai Pan, Rebecca Li, Zhiyu Dai; ¹Univ of Arizona, Phoenix, AZ,

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: We hypothesis that endothelial fatty acid metabolism is critical for obstructive vascular remodeling in the pathogenesis of PAH. Methods: Here we applied single-cell RNA sequencing (scRNA-seq) to profile the pulmonary cells in a severe mouse model (Egln1^Tie2Cre mice) of PH. Human hPAEC from idiopathic PAH patients and healthy donors were used to measure fatty acid-binding protein 4 and 5 (FABP4 and FABP5) expression. siRNA mediated knockdown of FABP4 and FABP5 was performed to study cell proliferation and apoptosis. Glycolysis assay was performed to evaluate the role of FABP4-5 on ECs. Egln1^Tie2Cre mice were bred with Fabp45^-/- mice to generate Egln1^Tie2Cre/Fabp45^-/- mice. Results: scRNA-seq analysis demonstrated that both FABP4 and 5 were highly induced in the ECs of Egln1^Tie2Cre mice. PAECs from IPAH patients also showed higher expression of FABP4 and 5. Knockdown of FABP4-5 reduced EC proliferation and starvation-induced Caspase 3/7 activity. Overexpression of FABP4-5 promoted EC glycolysis and proliferation. Genetic deletion of Fabp4 and 5 in Egln1^Tie2Cre mice exhibited a reduction of RVSP, RV hypertrophy, and reduction of EC glycolysis gene programming compared to Egln1^Tie2Cre mice. Conclusions: FABP4 and 5 control EC glycolysis and contribute to the development of PAH.

This research has received full or partial funding support from the American Heart Association.

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Key Words: Pulmonary hypertension; Endothelial function; Lipoproteins; Glucose

Kuang-yuh Chyu¹, Xiaoning Zhao², Jianchang Zhou³, Paul C Dimayuga¹, Wai Man Lio⁴, Bojan Cercek¹, Noah Trac⁵, Eun Ji Chung⁶, Prediman K Shah¹; ¹Cedars Sinai Med Cntr, Los Angeles, CA, ²Cedars Sinai Med Cntr, West Hollywood, CA, ³Cedars Sinai Med Cntr, W Hollywood, CA, ⁴Div of Cardiology, Cedars-Sinai Heart Institute, Cedars-Sinai Med Cntr, Los Angeles, CA, ⁵2Dept of Biomedical Engineering, Univ of Southern California, Los Angeles, CA, ⁶Univ of Southern California, Los Angeles, CA,

Introduction: ApoB-100 peptides provoke self-reactive immune responses in patients with atherosclerotic cardiovascular disease (ASCVD). It is unclear if there is a specific T cell memory population that responds to apoB-100 self-antigens and if it is modifiable. The use of self-peptides complexed to nanoparticles is under investigation as therapy to modulate immune self-reactivity in autoimmune disease. In this study, we assessed: 1) the intrinsic T Effector and T Effector Memory (TEM) response to the apoB-100 peptide P210 previously reported to generate self-reactive immune responses in ASCVD patients; and 2) the translational potential of P210-complexed to peptide amphiphile micelle (PAM) nanoparticles in reducing atherosclerosis in humanized Class-I MHC HLA-A*0201 chimeric mice on the apoE-/- background, developed in our laboratory. Methods and results: PBMCs from ACS patients (N=13) stimulated with P210 resulted in persistent CD8+ T Effector and TEM response, with no difference in CD4+ T cells, compared to controls (N=14; P<0.05). We then tested if P210-PAM nanoparticles altered the intrinsic T cell response to P210. We postulated if the intrinsic response to P210 was not altered by P210-PAMs, then the T cell responses to P210 peptide alone should correlate with the T cell response to P210-PAM. CD4+ T cell response to P210 peptide and P210-PAM were correlated (Spearman r=0.94; P=0.02; N=6) but CD8+ T cell responses were not, suggesting P210 complexed to PAM may alter its immune reactivity with CD8. The potential for P210-PAM as immune-modulating therapy was tested in humanized hypercholesterolemic HLA-A*0201 mice which had similar immune profile, serum cholesterol and aortic atherosclerosis compared to non-transgenic littermates. P210-PAMs were administered in 3 subcutaneous injections 2-3 weeks apart in mice of both sexes. A mouse serum albumin (MSA) peptide served as control. There was significantly reduced aortic atherosclerosis (P<0.05) in mice treated with P210-PAM (N=13) compared to PBS-treated mice (N=14) and those treated with MSA-PAM (N=20). Conclusion: The results suggest that nanoparticle-based immune modulation of T cell responses to apoB-100 may have translational therapeutic potential against atherosclerosis in ASCVD.

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Key Words: Atherosclerosis; Immunology; Immune system

Jeanne A Ishimwe¹, Evan C Ray², Thomas R Kleyman³, Annet Kirabo⁴, Cheryl L Laffer⁵, Alp Ikizler⁶, Fernando Elijovich⁷; ¹Vanderbilt Univ, VUMC, Nashville, TN, ²UPMC DIVISION OF NEPHROLOGY, Pittsburgh, PA, ³UNIVERSITY PITTSBURGH, Pittsburgh, PA, ⁴Vanderbilt Univ Med Cente, Nashville, TN, ⁵VANDERBILT U SCHOOL of MEDICINE, Nashville, TN, ⁶Nashville, TN, ⁷VANDERBILT UNIVERSITY, Nashville, TN,

Studies on the impact of sex as a biological variable on salt sensitivity of blood pressure (SSBP) are controversial. While population studies show that women are more salt-sensitive than men, studies in rodents indicate the opposite. Epithelial sodium channel (ENaC) plays an important role in SSBP. Dendritic cells sense Na⁺ through ENaC leading to formation of Isolevuglandins and promoting inflammation and hypertension. The mechanistic interplay between excess dietary Na⁺ and the role of sex as a biological variable in SSBP is still yet to be defined. Arachidonic acid (AA) and its metabolites including Epoxyeicosatrienoic acids (EETs) play an important role in SSBP, the latter by regulating the activity of ENaC. We hypothesized that regulation of ENaC by EETs contributes to sex differences in SSBP. We performed plasma metabolomics analysis in volunteers whose blood pressure and Na⁺ intake were monitored. We classified Na⁺ intake <2.3g as normal salt, and ≥ 2.3g Na⁺ as high salt, based on the American Heart Association recommendations. We found that women (n=81) had a stronger relationship between BP and Na⁺ intake than men (n=49) (r=0.372; p<0.001 vs. r=0.317; p=0.026). Among the participants consuming high Na⁺, women (28) had higher levels of AA than men (25), (1.119 ± 0.242 vs. 0.965 ± 0.201; p=0.015) and 12-HETE (1.329± 0.925 vs. 0.902 ± 0.520; p=0.047). Using the Weinberger protocol to assess salt sensitivity in humans, we found that increased urine EETs were associated with greater reductions of isolevuglandins in human antigen-presenting cells following salt depletion. In a novel mouse strain, the W521R mouse that carries a gain-of-function mutation in ENaC, we found that males developed high salt-induced hypertension when fed a HS diet for 3 weeks compared to baseline (134.3±3.7 mmHg vs. 111.9±4.8 mmHg, p=0.01) which was not observed in their littermate controls (119.3±5.4mmHg vs. 108.8±3.7mmHg, p=0.162). No blood pressure changes were observed in female W521R mice (102.3± 5.23 vs. 109.5± 7.75, p= 0.467) or littermate controls (99.5± 1.87 vs. 105.3± 4.95, p=0. 0.296). Our findings further indicate a paradoxical effect of sex on SSBP in humans and mice and suggest a role of ENaC regulation by AA metabolites.

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Key Words: Sex differences; Hypertension:experimental

Chun Ka Wong, Yee-Man Lau, Kevin Lai, Ricky Ruiqi Zhang, Hayes Kam hei Luk, Antonio Cheuk Pui Wong, Patrick Chiu Yat Woo, Susanna Kar Pui Lau, Kwok Hung Chan, Ivan FN HUNG, Chung Wah Siu; ¹Univ of Hong Kong, Hong Kong, Hong Kong

Background: Amid COVID-19 pandemic, there have been concerns regarding safety of angiotensin-converting enzyme inhibitor (ACEi) and sodium-glucose cotransporter 2 inhibitor (SGLT2i). Methods and Results: We evaluated the effects of ACEi and SGLT2i pretreatment on human induced-pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) infected by SARS-CoV-2. HiPSC-CM were pretreated with ACEi or SGLT2i for 7 days before SARS-CoV-2 infection. Three days after infection, viral replication was evidenced by increased supernatant SARS-CoV-2 RNA and presence of SARS-CoV-2 nucleocaspid protein within hiPSC-CM. Infected hiPSC-CM ceased spontaneous beating, exhibited cytopathic changes and had reduced viability. SARS-CoV-2 infection downregulated ACE2 while upregulated brain natriuretic peptides NPPB and proinflammatory chemokines CXCL1 and CXCL2 mRNA expression. ACEi pretreatment upregulated ACE2 expression by 5.87-fold (p<0.001***). Nonetheless, the increased ACE2 expression did not enhance SARS-CoV-2 infectivity. On the contrary, ACEi modestly improved hiPSC-CM viability by 12.1% (p<0.05^#) and down-regulated CXCL1 by from 69.2-fold to 27.0-fold (p<0.05^#). On the other hand, SGLT2i modestly reduced SARS-CoV-2 susceptibility with a reduction of supernatant SARS-CoV-2 RNA by 0.54 Log₁₀ copies/ml (p<0.05^#) and markedly downregulated CXCL1 and CXCL2 expression from 117-fold to 34.7-fold and from 166-fold to 110-fold respectively (p<0.05^#). Conclusion: Our findings contributed to a better understanding of ACE2 physiology in human hearts and anti-inflammatory effects of ACEi and SGLT2i on the myocardium.

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Key Words: COVID-19; Myocarditis; Sodium glucose co-transporter 2 inhibitors; Angiotensin II; Stem cells

Young Chun, Matthew Miyamoto, Charles Hong

Univ of Maryland Baltimore, Baltimore, MD,

Congenital dilated cardiomyopathy (cDCM) is a rare disease with unknown etiology, thus challenging therapeutic development. Here, we describe an infant with fulminant cDCM whose impaired myocardial contractility, and mitochondrial and sarcomere defects were modeled using induced pluripotent stem cell (iPSC) derived cardiomyocytes (CM). Whole-exome sequencing of the proband and his parents identified compound heterozygous mutations in the centrosomal protein rotatin (RTTN), which was confirmed as the defect responsible for the cDCM through genome editing and extensive functional testing. Our scRNA-sequencing analysis determined that severe dysregulation of CM contractile, mitochondrial, and metabolic genes in cDCM-CMs, as well as impaired cardiomyocyte maturation underlay the phenotypic defects. Mutations in RTTN in cDCM-CMs disrupted centrosome reduction, a developmentally programmed feature of CM maturation, resulting in persistent localization of the pericentriolar material at the centriole. Consequentially, it produced global defects in the microtubule network and disruption of mitochondrial and sarcomere development. We then identified a novel small molecule which restored centrosome reduction in cDCM-CMs. Interestingly, its treatment significantly restored comprehensive cell biology of cDCM-CMs, suggesting that centrosome reduction is a required step for CM maturation to occur and pointing towards a potential therapeutic for centrosome-mediated immature cDCM. Taken together, our data show that RTTN plays a crucial role in developmentally programmed CM centrosome reduction and identifies a potential therapeutic to restore proper cardiomyocyte structure and function in cDCM.

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Key Words: Cardiovascular disease

Lili Zhang¹, Jie Lv², Sen Zhu², Kaifu Chen¹; ¹Boston Children's Hosp, Boston, MA, ²Houston Methodist Rsch Institute, Houston, TX,

Background: Endothelial cell (EC) hold great promise for therapy in many diseases, thus understanding the molecular program governing EC phenotype and function will allow us to better treat cardiovascular pathologies. Our previous study had revealed that ITF2 is a novel cell identity gene for endothelial cells. The aim was to investigate the role of ITF2 in regulating EC phenotype and function. Methods: Standard EC functional assay (EC proliferation, migration, tube formation and nitric oxide (NO) production) were performed through ITF2 gain- and loss-of function approaches. Bulk RNA-Seq, scRNA-seq and ChIP-Seq were performed to dissect the pathways regulated by ITF2 in EC. Results: Epigenetic landscapes nominate ITF2 as a master regulator of EC identity. ITF2 knockdown (KD) significantly impaired EC function by decreasing EC cell proliferation, migration, tube formation and NO production. RNA-seq analysis showed that, ITF2 KD down-regulated genes were enriched in endothelial related pathways, while ITF2 KD up-regulated genes were highly enriched in Endothelial-Mesenchymal Transition (EndoMT) and TGFβ signaling pathway. In vitro experiment further confirmed that ITF2 KD in EC induced loss of EC characteristics and gain of fibroblast phenotype. Single-cell RNA-Seq analysis further revealed that ITF2 expression level could determine the extent of EndoMT in different EC subpopulations. Our ITF2 ChIP-seq data found that ITF2 showed strong binding peak on the promoter region of TGFβ1, TGFβR1 and Snail1, which indicate that ITF2 oppose EndoMT by negatively regulating TGFβ pathway. ITF2 overexpression in EC rescued EC dysfunction and EndoMT process induced by TGFβ1 stimulation. Conclusion: Taken together, our study elucidates a novel role for ITF2 in maintaining EC phenotypes and preventing EndoMT induced by TGFβ. As ITF2 overexpression could rescue EC dysfunction and oppose EC undergoing EndoMT, ITF2 may serve as a therapy target for EndoMT related disease.

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Key Words: Endothelial; Epigenetics

Hung-Chih Chen¹, Yen-wen Liu², Kuan-Cheng Chang³, Yen-Wen Wu⁴, Yu-Kai Chao¹, Min-Yi Yu¹, Riley Puntney, Chen-Ju Lin¹, Marvin L. Hsieh, Yu-Che Cheng⁵, Yi-Ming Chen¹, Jennifer Coonen⁶, Jia-Jung Lee⁷, Deng-Chyang Wu⁷, Ming-Jing Hwang¹, Chi-Liang Eric Yen, Federico Rey⁸, Timothy A Hacker⁹, Timothy J Kamp¹⁰, Patrick C Hsieh¹¹; ¹Academia Sinica, Taipei, Taiwan, ²Div of Cardiology, Tainan, Taiwan, ³China Med Univ & Hosp, Taichung City, Taiwan, ⁴Far Eastern Memorial Hosp, New Taipei City, Taiwan, ⁵Taipei, ⁶Wisconsin National Primate Rsch Cntr, Madison, WI, ⁷Kaohsiung Med Univ & Hosp, Kaohsiung, Taiwan, ⁸Madison, WI, ⁹Univ of Wisconsin, Madison, WI, ¹⁰U of Wisconsin-Madison, Madison, WI, ¹¹Academia Sinica, Taipei City, Taiwan

Background: The contribution of the gut microbiota and its metabolites to the development of heart failure after injury has been demonstrated. Advances in mechanistic understanding of the role of gut microbiota may lead to new prevention strategies and treatments for heart failure. In this study, we explored the gut microbiota and plasma metabolomes enriched in patients with ST-elevation myocardial infarction (STEMI), recapitulated the results in a non-human primate model and determined the post-MI metabolic regulation in mice using human fecal microbial transplantation. Methods: We recruited 64 controls and 64 coronary angiogram-confirmed STEMI patients, and collected their stool and plasma right after percutaneous coronary intervention (PCI, STEMIT1) and at 28 days after PCI (STEMIT2). We applied 16S rRNA NGS and shotgun metagenomics to reveal the gut microbiota in the control and patient samples. Moreover, we used NMR and LC-MSmetabolomics to determine the plasma metabolomic profiles. To test the causal effects, we treated germ-free mice with human fecal microbiota and SPF mice with candidate metabolites and examined their impact on mice post-MI. Furthermore, we tested if the human microbiomic and metabolomic results could be recapitulated in rhesus macaques with coronary ischemia-reperfusion (IR) injury. Results: Through 16S rRNA NGS and shotgun metagenomic analysis, we identified an enrichment of several butyrate metabolism-related bacteria in STEMI patients compared to the control. Germ-free mice receiving fecal microbiome transplantation of STMEI samples had reduced cardiac ejection fraction after MI. Moreover, NMR and LC-MS metabolomic analyses revealed an elevation in the biosynthesis and degradation of ketone bodies in the STEMI samples. Supplementation with butyrate, a source of ketone bodies, showed better cardiac protection in the presence of the gut microbiome. In rhesus macaques undergoing IR injury, we observed consistent elevation of gut butyrate metabolism-related bacteria and plasma ketone bodies after cardiac injury. Conclusion: This study demonstrates a correlation between gut butyrate metabolism-related bacteria and cardiac ischemic injury suggesting a novel role for keto-metabolism in cardiac repair.

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Key Words: STEMI; Cardioprotection; Microbiome; Metabolomics; Biomarkers

Brian R Weil, Alexandra K Gilligan, Dorcas Nsumbu, John M Canty, Jr.; ¹Univ at Buffalo, Buffalo, NY,

Introduction: We previously demonstrated that intracoronary cardiosphere-derived cells (CDCs) improve systolic function in swine with myocardial infarction and hibernating myocardium. In rats with hypertensive HFpEF, CDCs reduce interstitial fibrosis and improve diastolic left ventricular (LV) compliance. Whether these observations translate to a large animal model of HFpEF using an approach translatable to human disease is unknown. Methods: Swine (n=13) were subjected to daily (1 hr) episodes of repetitive LV pressure overload (RPO) for 2 weeks using phenylephrine (24 mg/hr) to transiently elevate LVEDP to ~30 mm Hg. We have previously demonstrated that this results in increased interstitial fibrosis and a two-fold reduction in LV diastolic compliance in the absence of anatomic LV hypertrophy. After 2 weeks, RPO was discontinued. Animals were started on cyclosporine (200 mg/day) and randomized to receive allogeneic CDCs (30x10⁶; n=7) or saline (n=6) into each of the 3 major coronary arteries and followed for an additional 4 weeks. Blinded LV end-diastolic volume index and EDP were measured at normal and elevated preload to assess LV compliance (ΔLVEDVi/ΔLVEDP). Results: Measurements of LVEDVi and LVEDP are summarized in the Figure. Two weeks of RPO decreased LV diastolic compliance from 0.47 ± 0.04 to 0.20 ± 0.03 mL/m²/mmHg (p<0.001). Four weeks after cessation of RPO, LV diastolic compliance remained impaired with no spontaneous recovery in saline controls (0.16 ± 0.03 mL/m²/mmHg). Likewise, LV compliance in CDC-treated animals was not different from measurements immediately before CDCs nor was it different from saline controls (0.15 ± 0.03 mL/m²/mmHg). Conclusion: These results demonstrate that (1) the impairment in LV compliance resulting from RPO persists without spontaneous improvement 4 weeks after cessation of RPO and (2) in contrast to hypertensive rats, intracoronary CDCs have no effect on LV diastolic compliance in swine with RPO-induced HFpEF.

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Key Words: Heart failure; Diastolic function; Cellular Therapy; Cardiovascular therapeutics; Hemodynamics

Itai M Magodoro¹, Carlos E Guerrero, Sr², Brian Claggett³, Stephen Jermy⁴, Hadil Saad⁴, Pablo Lamata⁵, Masliza Mahmod⁶, Jason V Baker⁷, Tess Peterson⁷, Landon Myer⁴, Heather Zar⁴, Mark Siedner⁸, Ntobeko Ntusi⁹, Goodarz Danaei¹, Mpiko Ntsekhe⁹; ¹Harvard Sch of Public Health, Boston, MA, ²Universidad de Cartagena, Cartagena, Colombia, ³Brigham and Women's Hosp, Boston, MA, ⁴Univ of Cape Town, Cape Town, South Africa, ⁵Kings Collège of London, London, United Kingdom, ⁶Univ of Oxford, Oxford, ⁷Univ of Minnesota, Minneapolis, MN, ⁸Harvard Med Sch, Boston, MA, ⁹Univ of Cape Town, Cape Town,

Background: Perinatally HIV infected (PHIV) adolescents are commonly surviving into adulthood due to antiretroviral therapy and maybe at increased risk of heart failure (HF) compared to uninfected peers. Hypothesis: We hypothesized that cardiac MRI- (CMR) may identify early cardiac changes among adolescent PHIV, and inform possible mechanisms and event risk. Methods: Seventy-one PHIV (mean age 15.2 years) and 36 age- and community-matched HIV-uninfected adolescents completed CMR including cine, T1 mapping and extracellular volume (ECV) estimation. Associations between HIV and LV remodeling indices were assessed. Results: PHIV adolescents had greater indexed LV end-diastolic volume (84.3 vs. 77.1 mL), sphericity index (0.41 vs. 0.38) and ECV fraction (29.1 vs. 28.0%), but smaller LV mass/volume ratio (0.68 vs. 0.75g/mL) vs controls (Table 1) . Increasing M/V ratio was accompanied by improved global circumferential strain (GCS) among controls (R=0.40; p=0.025) but not among adolescents with PHIV (R=0.13; p=0.514) (Fig. 1 and 2). Conclusion: Adolescents with PHIV have evidence of geometric, structural and functional LV remodeling. This remodeling is associated with myofibrosis and possible reduced reserve cardiac capacity suggesting a potential mechanism for increased HF risk in adulthood.

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Key Words: Fibrosis; Cardiac MRI; Acquired immunodeficiency syndrome; Heart failure, pediatric

Brandon Chalazan¹, Emma Leach¹, Krishnan Ramanthan¹, Matthew Bennet¹, Jag Walia¹, Laura Halperin¹, Julieta Lazarte², Robert A Hegele², Anna Lehman¹, Zachary Laksman¹; ¹Univ of British Columbia, Vancouver, Canada, ²Western Univ, London, Canada

Background: Atrial Fibrillation (AF) is the most common sustained cardiac arrhythmia worldwide. A substantial proportion of AF cases cannot be explained by acquired AF risk factors. Limited guidelines exist that support routine genetic testing. We aim to assess the clinical utility of genetic testing in patients with early- and very early-onset atrial fibrillation. Methods: From the literature, we identified 264 genes potentially related to AF, and then applied the ClinGen clinical validity framework to grade evidence supporting an association between specific genes and AF. Variants from exome sequencing in affected individuals were initially filtered at ≥20X read depth with genotype qualities ≥99%. A subsequent filter was applied to identify protein coding and splice site variants with minor allele frequencies ≤1.0% in gnomAD that were predicted to affect conserved residues and be damaging based on in-silico tools (GERP ≥3, SIFT ≤0.05, PolyPhen2 HDIV / HVAR ≥0.90, MutationTaster ≥0.50, CADD ≥20), prior to undergoing clinical classification for disease causation. Results: 200 AF individuals were ≤60 years of age and without any acquired AF risk factors at the time of AF diagnosis. The mean age of AF onset was 43.6 ±9.4 years, 167 (83.5%) were male, and 58 (29.0%) had a confirmed positive family history. We determined that 8 genes had sufficient evidence to be linked to an initial presentation of AF. From these AF-linked genes, only 4 (2.0%) individuals were identified to have a disease-causing variant. In addition, 55 (27.5%) individuals were found to have a variant of unknown significance. Lastly, we found that nearly half of the very early-onset AF (≤45 years of age) cohort carried the 4q25 at-risk SNP (rs6817105) compared to only 28.9% of early-onset AF (P=.01). Conclusions: This is the first study to provide a curated list of clinically actionable AF genes to genetically test in clinical practice, and highlights the opportunity for offering personalized management strategies in AF. This also highlights the use of clinical diagnostic criteria for identifying disease-causing variants in AF-linked genes. However, further work is still needed to dissect additional monogenic and polygenic determinants for AF.

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Key Words: Atrial fibrillation; Genetics; Genomics

Seshagiri Nandula¹, Sabyasachi Sen², Beda Brichacek³; ¹George Washington Univ, Washington, DC, ²Veterans Affairs Med Cntr, Bethesda, MD, ³The George Washington Univ, Washington, DC,

Introduction: COVID-19 attributed to SARS-CoV2 infection is a world-wide pandemic. SARS-CoV2 has been associated with cardiovascular disease and diabetes. Endothelial cell dysfunction (ECD)is quite common in type 2 diabetes mellitus (T2DM) where the renal podocyte dysfunction is often an early manifestation of microvascular complication. In this study we explored whether presence of hyperglycemia predisposes to increased SARS-CoV2 infection and whether co-morbid presence of COVID-19 and hyperglycemia predisposes to cardio-metabolic complications such as podocyte damage resulting in diabetic kidney disease (DKD). Methods: To estimate any possible kidney damage, in COVID-19, we evaluated albuminuria and podocyte markers in urine exosomes from SARS-CoV2 patients at 10 days, 6 months and 12 months post infection. Urine exosomes were isolated and identified using CD9, CD63 and Hsp-70. Podocyte protein markers such as Nephrin (Nep) and Podocalyxin (PODXL) were identified by western blot analysis. Results: HEK cells on exposure to hyperglycemia for 14 days showed upregulated expression of ACE2 and TMPRSS2 genes. HEK results prompted us to examine effect on podocytes in patients. All subjects with COVID had blood glucose levels above 300mg/dL therefore we used blood and urine samples from T2DM subjects (with no history of COVID-19) as a comparator. Mononuclear cells showed persistent over-expression of IL-6 and TNF-α (4-fold) even at 12 months post COVID. Urine-exosomal-protein Nephrin, (normalized to CD9 exosomal protein band intensities) was 10-fold higher (p=0.0003) compared to T2DM-Non COVID patients. Similar trend was observed with podocalyxin (PODXL) at 6 and 12 M (p=0.004) post-acute infection. Interestingly, there was no significant differences noted on urine albumin:creatinine (UAR) ratios between the two groups. Conclusions: Presence of hyperglycemia augments expression of ACE2 and TMPRSS2 mRNA expressions in HEK cells. Podocyte specific urinary exosomal estimation appears to be a more sensitive biomarker than UAR. A persistent high levels of podocyte specific proteins was noted in urinary exosomes even at 12 months post COVID indicating possible long-standing renal damage which may lead to renal failure and hypertension.

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Key Words: COVID-19; Diabetes (Type II); Inflammation and inflammatory markers; Kidney

Jenish Maharjan, Gina Barnes, Abigail Green Saxena, Jacob Calvert; ¹Dascena, Houston, TX,

Background: Ischemic stroke (IS), which accounts for ~87% of stroke cases, is one of the leading causes of mortality both in the US and globally. Effective primary and secondary preventative measures include anticoagulant therapy, which is also an effective treatment option for acute stroke. The development of new anticoagulant therapies is subject to safety and efficacy testing in clinical trials, which operate in a limited timeframe. Thus, identification of patient cohorts at high-risk for IS within a specific time window would maximize the impact of these trials. We hypothesized that machine learning algorithms (MLAs) could more accurately identify high-risk IS patients than traditional scoring systems. Methods: A retrospective study was performed using 58,179 qualifying inpatient encounters with IS from inpatient visits recorded from over 700 inpatient and ambulatory care sites. Patient data were extracted from electronic health records and used to train and test a gradient boosted MLA to predict the patients’ risk of experiencing IS from the period of one day up to one year following the patient encounter. The primary outcome of interest was the occurrence of IS. Results: The MLA obtained an AUROC of 0.876, and at a sensitivity of 80% obtained a specificity of 78.9% for prediction of IS within the next year. Features that significantly impacted the prediction of IS included previous stroke history, age and mean systolic blood pressure. Conclusions: MLAs can be utilized to more accurately predict the risk of IS within a 1-year window for hospitalized patients. This risk stratification tool could be used to design clinical trials to test anticoagulant stroke prevention therapies in high-risk populations by identifying subjects who would be more likely to benefit from treatment. Figure 1. Study design timeline and receiver operating characteristic (ROC) curve for the prediction of ischemic stroke for up to one year after inpatient encounter.

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Key Words: Stroke; Artificial Intelligence; Machine Learning; Clinical trials; Thrombolysis

YUKARI MASUDA¹, Naoya Yahagi², Yoshinori Takeuchi², Yuichi Aita², Yuki Murayama², Nobuhiro Wada², Yiren Huyan², Zahra Mehrazad Saber², Yoshikazu Sawada², Akito Shikama², Yoshihiko Izumida², Hitoshi Shimano³, Toshimasa Yamauchi¹; ¹Dept of Internal Medicine, Graduate Sch of Medicine, Univ of Tokyo, Tokyo, Japan, ²Nutrigenomics Rsch Group, Faculty of Medicine, Univ of Tsukuba, Tsukuba, Japan, ³Dept of Internal Med (Endocrinology and Metabolism), Faculty of Med, Univ of Tsukuba, Tsukuba, Japan

Introduction: Sterol regulatory element-binding proteins (SREBPs) are key transcription factors to regulate the de novo fatty acid and cholesterol synthesis. Between the two SREBP isoforms, SREBP-1 and -2, SREBP-1 mainly controls hepatic lipogenesis whereas SREBP-2 controls cholesterol synthesis. Previously we reported that PUFA selectively suppresses SREBP-1 activation by proteolytic processing, not affecting SREBP-2. However, the mechanism remains unclear. Methods: In previous report, we established an original reporter assay to assess the activities of SREBP proteolytic processing in vivo. To clarify the molecular mechanisms by which PUFA selectively suppresses SREBP-1, we tried to identify the PUFA-responsive domain on SREBP-1. For this purpose, we created various constructs of SREBP-1 and -2 chimeric molecules and measured the proteolytic processing activities of these molecules (Figures 1 and 2). Results: From these experiments, we were able to narrow down the PUFA-responsive domain to the C-terminal region of SREBP-1 (Figure 3). We have also found that this region is not involved in the interaction with SREBP cleavage-activating protein (SCAP). Conclusions: These results demonstrated that SREBP-1-specific PUFA response is independent of SCAP, suggesting that some adapter protein other than SCAP may be involved.

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Key Words: Fish oils; Nutrition; Cardiovascular; Healthcare innovation; Triglycerides

Ariel Bier¹, Eliyahu Shapira¹, rawan Khashab¹, Yehonatan Sharabi¹, Ehud Grossman², Avshalom Leibowitz¹; ¹Chaim Sheba Med Cntr, Ramat Gan, Israel, ²Chaim Sheba Med Cntr, Tel Hashomer, Israel

Introduction: A high fructose diet induces metabolic syndrome (MeS) which includes ectopic lipid accumulation, such as “fatty liver”. MeS is also associated with chronic kidney disease (CKD). A novel isoform of ChREBP, ChREBPβ, was recently reported to regulate liver fructose metabolism that leads to fatty liver development. The aim of this study was to evaluate fructose metabolism in the kidney and whether this metabolism leads to intra-renal fat accumulation. Methods: In-vivo: Sprague Dawley rats were fed either normal chow (Ctrl) or a high fructose diet (HFrD) for eight weeks. Blood pressure, fasting blood glucose and triglycerides were measured. The kidneys were harvested for ChREBPβ and de novo lipogenesis (DNL) gene expression, triglyceride content and histopathology staining.In-vitro: HK2 (human kidney) cells were treated with fructose for 48h and gene expression for ChREBPβ and DNL were determined. Results: The HFrD rats exhibited higher blood pressure (152 vs. 138 mmHg), glucose (146 vs. 127 mg/dl) and triglyceride (280 vs. 143 mg/dl) levels. Kidneys weight normalized to body weight of the HFrD rats were significant higher than the Ctrl (7.32 vs. 5.87). The difference can be explained by the higher triglyceride content in the HFrD kidneys (16.4 vs. 12.4 mg). Oil red staining revealed higher lipid droplet formation in the HFrD kidneys, which was also supported by higher adipophilin mRNA expression. The expression level of ChREBPβ and its downstream genes, scd and fasn, were elevated in the HFrD kidney. Treating HK2 cells with 40mM fructose increased ChREBPβ expression levels. Its downstream genes, fasn and acc, also showed an increasing trend. In three out of five experiments, adipophilin was increased as well. Conclusion: In this study, we demonstrated that fructose consumption leads to intra-renal lipid accumulation and to the formation of a “fatty kidney”. This suggests a potential mechanism that can partially explain CKD development in fructose induced MeS.

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Key Words: Kidney; Cardiorenal metabolic risk; Metabolic syndrome

Jenish Maharjan, Gina Barnes, Jacob Calvert, Qingqing Mao; ¹Dascena, Houston, TX,

Background: A pulmonary embolism (PE) is a life-threatening condition associated with high morbidity and mortality, with mortality rates around 30% in untreated PE and 8% in treated PE. Successful treatment outcomes are contingent upon early and accurate recognition of PE. However, current risk stratification tools for PE are limited and unable to accurately predict PE events before their occurrence. Objective: This research presents a machine learning algorithm (MLA) designed to identify patients at risk of PE prior to the clinical detection of onset in an inpatient population using the first 3 hours of required patient data derived from electronic health records (EHRs). This tool is designed to enable earlier PE diagnosis and more timely intervention and provide clinicians with the opportunity to improve patient outcomes. Methods: Three machine learning (ML) models were developed on electronic health record data from 63,798 inpatients in a large United States medical center. These models included logistic regression, neural network, and gradient boosted tree (XGBoost) models. All models used only routinely collected demographic, clinical, and laboratory information as inputs. All were evaluated for their ability to predict PE prior to the clinical detection of onset in an inpatient population. Performance was assessed with regard to area under the receiver operating characteristic (AUROC), sensitivity, and specificity. Results: The model trained using XGBoost demonstrated the strongest performance for predicting PEs, obtaining an AUROC of 0.85, sensitivity of 81%, and specificity of 70%. The neural network and logistic regression models obtained AUROCs of 0.71 and 0.61, sensitivity of 81% and 81%, and specificity of 44% and 35%, respectively. Several features were identified as important for model predictions, including major trauma, previous VTE, and changes in patient clotting time. Conclusions: This algorithm may improve patient outcomes through earlier recognition and prediction of patients at high risk for inpatient PE, enabling earlier confirmatory diagnostic testing and treatment of PE.

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Key Words: Anticoagulants; Thrombosis; Artificial Intelligence; Vascular disease; Embolism

Stanislovas Jankauskas¹, Jessica Gambardella², Urna Kansakar³, Fahimeh Varzideh³, Ayobami Adebayo⁴, Michael Eacobacci⁴, Kwame Donkor⁴, Scott Wilson³, Gaetano Santulli³; ¹Albert Einstein College of Medicine, Bronx, NY, ²Univ of Naples Federico II, Naples, Italy, ³AECOM - ITME, NYC, NY, ⁴

Introduction: We and others have shown that endothelial cells (ECs) play a pivotal role in COVID-19. In recent years mounting data demonstrated the importance of mitochondria in endothelial dysfunction. Methods: We obtained plasma samples of patients hospitalized with COVID-19 on the first day of hospital admission. Samples were then divided into survivors (patients dismissed from the hospital) and non-survivors. Plasma samples from healthy individuals were used as control. The study was approved by the local Ethical Committee. Human umbilical vein ECs were treated with plasma from each group for 24 hours. Results: Plasma from COVID-19 patients increased lipid peroxidation, impaired angiogenic capacity, and caused EC apoptosis. Nitric oxide (NO) production was not different between ECs treated with survival group plasma vs control plasma, but was significantly reduced in ECs treated with plasma from non-survivors. Similarly, mitochondrial potential was unchanged in cells incubated with plasma from survivors and healthy subjects, whereas plasma from non-survivors caused a robust decrease in mitochondrial potential. Mitochondrial uncoupler FCCP caused a ~1.5 bigger decrease in mitochondrial potential in cells incubated with plasma from non-survivors compared to plasma from survivors. Treatment with ATP synthase inhibitor oligomycin resulted in robust loss of mitochondrial potential in cells exposed to plasma from both survivors and non-survivors, with a significantly more pronounced degree of mitochondrial de-energization in the non-survival group. Strikingly, the decrease in mitochondrial potential - both before and after oligomycin - caused by plasma from non-survivors was rescued by co-incubation for 24 hours with cyclosporin A, an inhibitor of mitochondrial permeability transition pore, and L-Arginine. Moreover, L-Arginine restored NO production, diminished lipid peroxidation, and ameliorated EC angiogenic capacity. Conclusion: We demonstrated that plasma of COVID-19 patients causes severe mitochondrial dysfunction in ECs and severity of mitochondrial dysfunction significantly correlates with death caused by COVID-19.

This research has received full or partial funding support from the American Heart Association.

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Key Words: COVID-19; Endothelium

Colin Clark, Daniel Infield, Jason Dierdorff, Abagail McKernan, Barry London, Christopher A Ahern; ¹Univ of Iowa, Iowa City, IA,

The voltage-gated sodium channel Na_V1.5 controls cardiac excitability and is an established therapeutic target. Mutations in the SCN5A gene, which encodes Na_V1.5, are associated with inherited arrhythmia syndromes, including Brugada and Long-QT. To advance the general understanding of Na_V1.5-related conduction biology, we have developed a chemical-genetic model to achieve acute and reversible silencing of Na_V1.5 in vitro and in vivo. To this end, a human Na_V1.5 chimeric channel was engineered to contain a high-affinity, isoform-specific binding site for acylsulfonamide (GX) drugs. The GX drug binding site is comprised of an extracellular-facing pocket formed by the DIV voltage-sensor (VSD4) thus enabling a structure-based chimera design strategy. The Na_V1.5-GX channel has WT voltage-dependent gating and, unlike WT Na_V1.5, is rapidly and reversibly inhibited by nanomolar GX compound. Using CRISPR, the GX binding site has been engineered into the homologous region of the endogenous Scn5a locus, thus phenocopying the chimeric construct. Inheritance of the Na_V1.5-GX allele follows expected Mendelian ratios, allowing for the production of a Na_V1.5^GX/GX homozygous strain. In the absence of GX compound, Na_V1.5^GX/GX hearts display normal cardiac phenotypes in vivo measured by EKG and echocardiography. Patch-clamped Na_V1.5^GX/GX sodium channels in isolated adult myocytes have WT gating but nanomolar GX compound ablates the Na_V1.5 mediated current. Acute exposure to GX compounds (GX-201, MRL5) in vivo by IP injection causes borderline QRS prolongation in Na_V1.5^GX/WT heterozygous mice, and marked PR and QRS prolongation, heart block, atrial arrhythmias and death in Na_V1.5^GX/GX homozygous mice. This powerful new resource will enable the critical assessment of the roles of sodium channel isoforms in cardiac function and disease progression, and will act as a test-bed for the evaluation of SCN5a patient mutations and their potential therapies.

This research has received full or partial funding support from the American Heart Association.

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Key Words: Cardiovascular; Sodium channel; Electrophysiology; Pharmacogenetics; Long QT syndrome

Mark A Sussman¹, Carolina Esquer², Clarissa Savko¹, Grant Shain¹, Oscar E Echeagaray¹, Fareheh Firouzi¹, Pria Bose¹, Abbie Rieder¹, Sophie Rokaw¹, Andrea Witon-Paulo¹, Natalie A Gude³; ¹San Diego State Univ, San Diego, CA, ²San Diego State Rsch Foundation, San Diego, CA, ³SDSU Rsch Foundation, San Diego, CA,

Vaping of flavored liquids has been touted as safe alternative to traditional cigarette smoking with decreased health risks. Popularity of vaping has dramatically increased over the last decade, particularly among teenagers who incorporate vaping into their daily life as a social activity. Moreover, inclusion of nicotine salts in vape juices renders them highly addictive leading to increased frequency of use and intractable addiction. Despite widespread and increasing adoption of vaping among young adults there is little information on long term consequences of vaping and potential health risks. Fundamental studies to determine the impact of vaping upon airway and lung tissue are desperately needed using translationally relevant animal models. This study demonstrates Vaping-Induced Pulmonary Injury (VAPI) using commercial JUUL pens with flavored vape juice in a translationally-relevant inhalation exposure murine model. Profound pathological changes to upper airway, lung tissue architecture, and cellular structure are evident within 9 weeks of exposure. Marked histologic changes include increased parenchyma tissue density, cellular infiltrates proximal to airway passages, alveolar rarefaction, increased collagen deposition, and bronchial thickening with elastin fiber disruption. Elevation of mucus secretion, inflammatory activity, and tissue remodeling are evident. Transcriptional reprogramming in vaped mice includes significant changes to gene families coding for xenobiotic response, glycerolipid metabolic processes, and oxidative stress revealed by spatial analyses. Cardiac contractile performance for systemic output is moderately but significantly impaired, and the pulmonary side shows severe structural remodeling with chamber enlargement indicative of right ventricular failure. Findings presented here document the first VAPI inhalation exposure model instigated using popular products sourced entirely from retail markets revealing mechanistic underpinnings of vaping-related pathologic injury leading to severe respiratory distress. Clinically-relevant studies to identify confounding factors and interventional mitigation approaches for VAPI-Acute Respiratory Distress Syndrome (ARDS) are underway.

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Key Words: Smoking; Pulmonary; Pulmonary heart disease; Cardiovascular disease

Bing Xu, Wuqiang Zhu; Mayo Clinic, Scottsdale, AZ,

Background: Impaired myocardial metabolism and energy supply are involved in the development of heart failure after acute myocardial infarction. We identified two cardioprotective chemicals, the fibroblast growth factor 1 (FGF1) and CHIR99021, that confer cardioprotection in the mouse and pig models of myocardial infarction. Here, we aim to determine if metabolic improvement contributes to FGF1 and CHIR99021 mediated cardioprotection. Methods: Myocardial infarction was surgically induced on 8-10 weeks old C57BL/6J mice. Nanofibers formulated with FGF1 and CHIR99021 was intramyocardially injected to three sites in the infarction area immediately after myocardial infarction induction. Animals were euthanized at day 3 and 7 after surgery and nanofiber treatment. Hearts were harvested for liquid chromatography tandem mass spectrometry-based metabolomics assays. Cell energy phonotype was determined by seahorse assay on isolated neonatal mouse cardiomyocytes treated with FGF1 (100ng/mL) and CHIR99021 (5µM). Results: Principal component analysis revealed a clear separation of metabolomic profile in the myocardium of sham and post myocardial infarction animals receiving treatment of empty nanofibers or nanofibers formulated with FGF1 and CHIR99021. Three days of ischemia resulted in increased the abundance of metabolites for glycolysis and glycogenolysis which were further enhanced by FGF1 and CHIR99021. Ischemia also increased the abundance of lactate but it was attenuated by FGF1 and CHIR99021. In addition, ischemia reduced the abundance of intermediates in tricarboxylic acid cycle which was not affected by FGF1 and CHIR99021. The impact of FGF1 and HICR99021 on glycolysis was lost seven days post-MI. FGF1 and CHIR99021 had no significant impact on lipid and amino acid metabolism at either three or seven days post myocardial infarction. Conclusion: FGF1 and CHIR99021 transiently improve the capability of ischemic myocardium to utilize glycolysis as energy resource without lactic acidosis.

This research has received full or partial funding support from the American Heart Association.

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Key Words: Myocardial infarction; Metabolism; Metabolomics; Cardioprotection

Karel T Breeman¹, Bryan J Swackhamer², Amy Brisben³, Anne-Floor B Quast¹, Allan Shuros³, Brian Soltis³, Brendan Koop³, Martin C Burke⁴, Arthur A Wilde¹, Fleur V Tjong¹, Reinoud E Knops¹; ¹Amsterdam UMC, Univ of Amsterdam, Amsterdam, Netherlands, ²Boston Scientific, St Paul, MN, ³Boston Scientific, St. Paul, MN, ⁴CorVita Science Foundation, Chicago, IL,

Introduction: Subcutaneous ICDs (S-ICDs) and leadless pacemakers (LPs) were designed to diminish complications related to transvenous leads. As S-ICDs do not deliver antibradycardia or antitachycardia pacing (ATP), no coordinated leadless option is available for patients with defibrillator and (expected) pacing needs. We preclinically evaluated system performance and safety of the first modular cardiac rhythm management (mCRM) system: a wirelessly communicating ATP-enabled LP (EMPOWER™) and S-ICD. Methods: An LP was implanted in 63 canine subjects and in 38 a concomitant S-ICD was implanted. Subjects were evaluated at multiple follow-up visits up to 18 months. At all visits, mCRM communication thresholds (CTs) (3 postures; successful if ≤6.0V post-implant and maximum 1.0V higher at later visits), LP electrical parameters and system complications were assessed. Performance stability was assessed from 7 days to 18 months (after subacute phase). Results: mCRM communication was successful in 1022/1024 (99.8%) attempts throughout the study, of which 45/45 (100%) at 18 months. Median CTs were 2.0V (IQR 1.5-2.0) at implant and were stable afterwards (median 1.5V, IQR 1.0-2.0) (Fig. 1A). At implant, pacing capture threshold (Fig. 1B), impedance and R-wave amplitude were median [email protected] (IQR 0.2-0.3), 877Ω (IQR 739-1041) and 25mV (IQR 14-34), respectively. Pacing thresholds remained stable (median [email protected]; IQR 0.1-0.9) and impedance (median 765Ω; IQR 578-953) and R-wave amplitudes (median 25 mV; IQR 13-37) were in acceptable ranges. Two system complications occurred, both were prototype LP accelerometer malfunctions. Conclusions: The first mCRM system has excellent performance up to 18 months with low, stable device-device communication thresholds and acceptable, stable LP electrical parameters. Long-term safety was acceptable as all complications were due to prototype LPs.

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Key Words: Pacemakers; Defibrillator; Pacing; Sudden cardiac death

Asma Boukhalfa¹, Sarah Powers¹, Dawn Meola², Greg Martin¹, Yuan Hushan³, Robert Blanton¹, Vicky Yang⁴, Gordon Huggins¹, Chen Howard¹; ¹MCRI Tufts Med Cntr, Boston, MA, ²Tufts Univ, North Grafton, MA, ³Gordon Cntr for Med Imaging, Massachusetts General Hosp, Boston, MA, Boston, MA, ⁴: Cummings Sch of Veterinary Medicine, Tufts Univ, Grafton, MA, Grafton, MA,

Introduction: Current molecular understanding of Hypertrophic Cardiomyopathy (HCM) remains incomplete and warrants further investigation. We previously demonstrated that autophagy required to maintain intracellular homeostasis is perturbed in HCM patient myectomy samples and in murine models carrying the formin protein FHOD3-V1151I genetic variant. We hypothesize that abnormalities in cardiomyocyte autophagy leads to dysregulated intracellular vesicular transport and extracellular vesicles secretion. Methods: Extracellular vesicles, specifically 30-100 nm exosomes, were purified from HCM patient plasma samples and from FHOD3-V1151I variant mice by chromatography. Physical properties of exosomes were characterized by dynamic light scattering, and composition by protein blots of LC3, CD9, ALIX and Troponin. To investigate the pathophysiological impact, pressure overload was induced in mice by transverse aortic construction (TAC), cardiac function was assessed by echo, and heart and plasma samples were analyzed. Results: In FHOD3-V1151I mice (n=4), exosomal proteins CD9 and ALIX were significantly downregulated compared to littermate controls (n=4), reflecting reduced circulating exosomes. Exosome volume and surface charge also differed significantly, suggesting altered compositions. Rapamycin injection (2 mg/kg i.p.) in mice restored autophagy flux (LC3-II) and exosome secretion (CD9/ALIX). Conversely autophagy inhibition by chloroquine (10 mg/kg i.p.) exacerbated the blockade of exosome secretion. Upon TAC surgery for 4 days, FHOD3-V1151I mice (n=6) exhibited significant 2-fold reduction in circulating exosomes compared to littermate control TAC mice (n=6). Ejection fraction was preserved but on day 28 worsened in FHOD3-V1151I TAC mice versus controls. Exosomes from FHOD3-V1151I TAC mice on day 4 had a 2.5-fold enrichment in cardiac Troponin T levels, confirming cardiomyocyte origin. Similar troponin elevation and CD9/ALIX reduction were seen in plasma exosomes from HCM human patient samples with the FHOD3-V1151I genotype. Conclusions: The cardiac autophagy and circulating exosome perturbations, reversible by autophagy modulation, raise intriguing possibilities in the early diagnosis and treatment of HCM

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Key Words: Autophagy; Hypertrophic cardiomyopathy; Cardioprotection; Heart failure; Biomarkers

Yijun Yang¹, Eric Feldsott², Alexander Hobby³, Giana Schena¹, Jaslyn Johnson⁴, Tao Wang², Thomas E Sharp⁵, Remus Berretta⁶, Joanne Donnelly⁷, Christopher Haydel⁷, Eric T Choi⁷, Sadia Mohsin⁸, Steven R Houser⁹, Hajime KUBO²; ¹Philadelphia, PA, ²TEMPLE UNIVERSITY, Philadelphia, PA, ³Univ of Colorado, Englewood, CO, ⁴Lewis Katz Sch of Medicine, Philadelphia, PA, ⁵Temple Univ Sch of Medicine, Philadelphia, PA, ⁶Temple Med Sch, Philadelphia, PA, ⁷TEMPLE UNIVERSITY HOSPITAL, Philadelphia, PA, ⁸Temple Univ, Philadelphia, PA, ⁹iMMEDIATE PAST President- AHA, Philadelphia, PA,

We have previously identified a cell type derived from mouse and pig cortical bone stroma that, when injected into the border zone after a myocardial infarction (MI), improves post MI remodeling. In the present research, we isolated and explored potential use of the human version of these bone stromal cells, which we have termed Immune Modulating Paracrine Acting Cells (IMPACs) based on their immune moderating characteristics, for cell therapy targeting post MI repair. IMPAC expressed and secreted immune modulatory factors IL-4, IL-10, IL-1RN, and TGFb. In-vitro studies showed that the conditioned medium derived from IMPACs had potent anti-inflammatory effects, suppressing IL-6 production in human macrophages and endothelial cells stimulated by inflammatory cytokines; TNFa, IL-1b, and INFg. When injected into border zones of a mouse model of MI, IMPACs were still observed in the MI border zone at the end of the 42-day experimental period. Their presence was associated with improvement in the cardiac functions, myocardial scar size, and level of dilation. The post MI tissue analysis revealed that the IMPACs treated animals had reduced neutrophil infiltration into the infarcted area and alterations in the phenotype of immune cell subset towards a reparative phenotype. Both early appearance and receding of alpha smooth muscle actin positive myofibroblasts was found, indicating that the IMPAC treatment promoted early initiation and completion of reparative phase. These beneficial effects could be due to the abundant amount of Thrombospondin 1 (TSP1) secreted by IMPACs that TSP1 is known to regulate repair through activation of TGFb; a master regulator of the transition from post MI inflammation to a reparative phase after myocardial injury. TSP1 signaling also suppressed degradation of damaged tissue through inhibition of matrix metalloproteinases contributing to preservation of the cardiac structure and cardiac remodeling. Collectively these studies show that IMPACs have the ability to suppress overly activated post MI inflammatory responses and promote repair, resulting improved cardiac structure and functions. IMPAC’s ability to potentially evade immune cells and survive within host myocardium for a long period of time is a beneficial quality.

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Key Words: Stem cell therapy; Stem cells; Myocardial infarction; Regenerative medicine stem cells

Lale A Ertuglu¹, Cheryl L Laffer², Fernando Elijovich³, Melis Sahinoz⁴, Ashley Pitzer⁴, Alp Ikizler⁵, Annet Kirabo⁴; ¹Vanderbilt Univ Med Cntr, Nashville, TN, ²VANDERBILT U SCHOOL of MEDICINE, Nashville, TN, ³VANDERBILT UNIVERSITY, Nashville, TN, ⁴Vanderbilt Univ Med Cente, Nashville, TN, ⁵Nashville, TN,

We showed that antigen-presenting cells (APCs) have a membrane ENaC. Na+ entry via this channel activates NADPH oxidase and formation of isolevuglandins (isoLG) that adduct to proteins, generating neoantigens that trigger inflammation in hypertension. This response is variable in human APCs. It is not known if this variability relates to individual salt sensitivity of blood pressure and whether it is produced by different individual regulation of function of the channel.We measured systolic blood pressure (SBP), isoLG-containing dendritic cells (DC), CD14+16-, CD14-16+ and CD14+16+ monocytes (flow cytometry and a specific antibody), plasma aldosterone (Aldo), and plasma and urine epoxyeicosatrienoic acids (PEETs and UEETs, the sum of the 8-9, 11-12 and 14-15 isoforms by HPLC) in 9 hypertensive subjects who were off therapy for 2 weeks, before (B) and after in-patient 24 hr salt loading (HI, 460 mmol Na+) and salt depletion (LO, 10 mmol Na+/24 plus furosemide 40 mg x 3).Age was 51±2, with 33% female, BMI 34.5±3.8 kg/m² and screening SBP 139±5 mmHg mmHg. Urine Na⁺ excretion was 182±18 in B, 378±27 in HI, and 28±3 mmol in LO after furosemide. SBP responses to salt loading varied from -4.7 to 9.1 mmHg and those to salt-depletion from +5.6 to -13.8 to mmHg. isoLGs were not different among B, HI or LO in any cell type. In contrast, ΔisoLGs produced by combined HI and LO in DCs (not monocytes) correlated with ΔSBP (r=0.70, p<0.002), whereas those of monocytes (not DCs) correlated negatively with ΔUEETs (r=-0.58 to -0.77, p<0.02 to <0.0005). However, in bivariate analyses both ΔSBP and ΔUEET were significant regressors for ΔisoLGs in the four cell types, explaining 55 to 89% of their variability. There were no correlations between PEETs or Aldo and isoLGs in any cell type.We conclude: (1) ENaC of APCs is not regulated by Aldo or systemic PEETs, and (2) Within the framework of our hypothesis, the bivariate analyses suggest that (a) Renal EETs inhibit APC ENaC as they do renal ENaC, thus regulating isoLG generation by Na+, (b) the effect may reflect exposure of APCs to Na+ in the renal medulla (relationships with UEETs but not PEETs) and (c) the resulting renal EET-regulated change in isoLGs by changes in Na+ balance is associated with the degree of salt sensitivity of blood pressure in humans.

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Key Words: Immune system; Hypertension; essential; Immunology; Sodium channel; Isoeicosanoids

silvie suriany¹, Honglei Liu², Andrew L Cheng², Christopher Denton³, Sarah Badran², Neil Patel², Thomas Coates³, John Wood⁴, Jon A Detterich²; ¹Div of Cardiology, Children's Hosp Los Angeles, Los angeles, CA, ²Div of Cardiology, Children's Hosp Los Angeles, Los Angeles, CA, ³Hematology Section, Cancer and Blood Disease Institute, Children's Hosp Los Angeles, Los Angeles, CA, ⁴Div of Cardiology, Children's Hosp Los Angeles,, Los Angeles, CA,

Introduction: Fontan palliation results in passive blood flow to the lungs; thus, viscoelastic properties of blood are a primary determinant of pulmonary vascular resistance. We aimed to determine whether viscoelastic properties of blood, specifically red blood cell aggregation (a determinant of low-shear blood viscosity), are different in Fontan patients vs. healthy controls. Methods: Whole blood viscosity was measured at native and varying hematocrits using cone-plate viscometry and tube viscometry over various shear rates in healthy controls and patients with Glenn and Fontan circulations. Red blood cell (RBC) deformability and aggregation were measured using couette and cone-plate aggregometers. Aggregation was also measured after RBC-plasma switching between compatible healthy and Glenn/Fontan blood. Results: Blood viscosity at native hematocrit was not different between Fontan (FN) [n=28], Glenn (GL) [n=12] and healthy [n=22] groups at any shear rate using tube viscometry. Hematocrit-to-viscosity ratio (HVR), a marker of microcirculatory oxygen delivery potential, was higher/improved in FN and GL groups vs. controls across all hematocrits and shear rates (p<0.0001); FN and GL HVRs were similar. RBC aggregation at low shear were lower in GL (13.98±1.17 a.u.) and FN (17.35±0.89 a.u.) groups vs. controls (22.03±0.85 a.u.) (p<0.0001). Suspending RBCs from GL patients in healthy plasma resulted in increased RBC aggregation at stasis (+5.058±1.56 a.u., p=0.0317) and low shear (+4.48±1.9 a.u., p=0.0793). Similarly, healthy plasma caused increased RBC aggregation in FN patients at stasis (+5.87±1.86 a.u., p=0.0159) and low shear (+6.29±2 a.u., p=0.0161). Correspondingly, FN and GL plasma lowered healthy RBC aggregation at stasis (-6.39±0.98 a.u., p<0.0001 and -7.74±1.5 a.u., p=0.0004) and low shear (-6.31±0.93 a.u., p<0.0001 and -6.37±0.87 a.u., p<0.0001). Conclusion: RBC aggregation is decreased in Fontan and Glenn circulations, but is increased by RBC suspension in plasma from healthy donors. Decreasing aggregation, and thus low shear rate viscosity, is a physiological compensation that may be a target for improvement of vascular resistance in patients with passive pulmonary circulations.

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Key Words: Fontan physiology; Single ventricle; Hemodynamics

Chen-Ju Lin¹, Yu-Che Cheng², Hung-Chih Chen³, Yu-Kai Chao¹, Patrick C Hsieh¹; ¹Academia Sinica, Taipei City, Taiwan, ²Taipei, ³Academia Sinica, Taipei,

Background: The gut microbiota plays a vital role in maintaining tissue homeostasis and regulating disease pathophysiology. Dysbiosis has been associated with cardiovascular disorders but the underlying mechanisms remain largely unexplored. We previously reported that mice depleted of gut microbiota were prone to cardiac rupture, suggesting that the gut microbiota is involved in the structural remodeling after heart injury. Here, we aimed to determine whether the gut microbiota modulates cardiac mechanics in health and under mechanical stress. Methods: We performed transverse aortic constriction (TAC) in mice treated with antibiotics to deplete the gut microbiota (ABX mice). Cardiac function was evaluated by echocardiography and catheterization, followed by heart tensile test and extracellular matrix (ECM) studies (n≥6 for each group). We further tested the effect in germ-free mice by transplanting cecal contents from healthy or diseased donors (n≥5 for each group). To identify the difference between these two microbial communities, we applied full-length 16S rDNA sequencing and used PICRUSt2 analysis to predict the metabolic pathways. Finally, to test the efficacy of metabolites enriched in healthy donors, we supplemented ABX mice with the metabolites (n≥6 for each group) and evaluated its function in boosting ECM homeostasis. Results: Cardiac hypertrophy and dysfunction were more severe in ABX mice after TAC compared to the control. Moreover, hearts of ABX-treated mice were less elastic and tough. Histological analysis confirmed that TAC-induced cardiac fibrosis was more severe in these mice, accompanied by disrupted cardiac ECM structure. Reconstruction of gut microbiota from healthy donors in ABX mice and germ-free mice successfully restored cardiac function and prevented ECM disarray. Furthermore, functional prediction identified shortchain fatty acids (SCFAs) as critical mediators in mechanical stress. Finally, supplementation of SCFAs improved cardiac function and reversed ECM disarray after TAC. Conclusion: This study implicates the gut microbiota as a potential therapeutic target for modulating cardiac ECM deposition and structural integrity under pressure overload stress.

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Key Words: Cardiovascular disease; Microbiome; Hypertrophy

Yu-Che Cheng¹, Marvin L. Hsieh², Chen-Ju Lin¹, Ching-Ying Huang¹, Cindy M.C. Chang², Riley Puntney², Martin W. Nicholson¹, Hung-Chih Chen¹, Gina C. Kim², Jennifer Coonen², Yen-wen Liu³, Timothy A Hacker⁴, Timothy J Kamp², Patrick C Hsieh⁵; ¹Academia Sinica, Taipei, Taiwan, ²Univ of Wisconsin-Madison, Madison, WI, ³Div of Cardiology, Tainan, Taiwan, ⁴Univ of Wisconsin, Madison, WI, ⁵Academia Sinica, Taipei City, Taiwan

Background: Cell therapy using pluripotent stem cell-derived cardiomyocytes to remuscularize the post-infarct myocardium has been reported. However, the relative immaturity of transplanted cardiomyocytes and poor survival rates due to limited blood supply are still major hurdles to successful outcomes. We tested if coculture of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) with hiPSC-derived endothelial cells (ECs) would promote CM maturation in vitro, and if cotransplantation of both hiPSC-CMs and hiPSC-ECs may facilitate hiPSC-CM muscularization in mice with myocardial infarction (MI) and in healthy non-human primates. Methods and Results: We generated three lines of hiPSC-CMs and hiPSC-ECs from three independent donors and determined that coculturing with hiPSC-ECs promoted the maturation of sarcomere and gap junction structures and the electrophysiology of hiPSC-CMs in a direct cell-cell contact manner. Next, we examined the therapeutic effect of cotransplantation of hiPSC-ECs and hiPSC-CMs in NOD-SCID mice undergoing MI. These mice showed a significant increase in left ventricle ejection fraction post-MI, compared to low or high doses of hiPSC-CMs alone or hiPSC-ECs alone (n≥14). Fewer arrhythmic events were observed in mice receiving cell cotransplantation. In healthy rhesus macaques (n=4), we demonstrated successful engraftment, maturation and integration of hiPSC-CMs with the host myocardium by cotransplantation with hiPSC-ECs. Importantly, in both mice and non-humanprimates, we found that human grafts generated by cotransplantation showed a higher vascular density in the grafts, with some neovessels formed by hiPSC-ECs. We also found erythrocytes inside the new vessels, indicating a functional connection to the host coronary circulation. Conclusions: This study demonstrates the beneficial effects of cotransplantation of hiPSC-CMs and hiPSC-ECs, which appears to promote maturation of hiPSC-CMs and enhance neovascularization in mouse and non-human primate hearts. Delivery of this combination of cell types holds promise for future clinical cardiac regeneration.

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Key Words: Cardiac regeneration; Stem cell therapy; Myocardial revascularization; Arrhythmias, treatment of; Regenerative medicine stem cells

Zhu Jinyun¹, Ning Zhang²; ¹Zhe jiang Univ, Hangzhou, China, ²Zhejiang Univ, Hangzhou, China

Introduction: Recurrent myocardial ischemia is common in patients with coronary artery disease, which predicts adverse outcome in patients with unstable angina. Since HIF-1a is a major player in monocytes activation and targets epigenetic enzymes, we hypothesize that repeated ischemia could lead to “trained immunity” in macrophages. Methods: The recurrent transient ischemia model was induced by ligating the left anterior descending coronary artery for five min followed by reperfusion and rest one week. Mice were divided into trained and sham groups. The trained mice were subjected to once, twice and three times insult of ischemia. In vitro, monocytes from C57Bl6 mice were exposed to normoxia or hypoxia for 24h followed by a resting period of 3 days to differentiate to macrophages, then replaced to normoxia or hypoxia. Results: Recurrent ischemia triggers significant cardiac fibrosis and exacerbated cardiac dysfunction in mice compared with sham, once and twice ischemia insults. More macrophages (CD11b⁺Ly-6G⁻F4/80⁺) are in the recurrent ischemia myocardium, whereas there are no differences of neutrophils (CD11b⁺Ly-6G⁺) between groups. Intriguingly, cardiac macrophages are basically CCR2⁺ cells, suggesting that vast majority of them are derived from bone marrow. In vitro, hypoxia training results in increased mRNA level and production of proinflammatory cytokines (IL-6, TNF-α, and IL-1β) compared with normoxia and only once hypoxia insult. Hypoxia training in macrophages leads to activate mTOR-HIF1α pathway and increased ROS production. According to clinical characteristics, patients with recurrent angina have increased number of white blood count compared with their initial chest pain. A positive correlation between peripheral leukocyte count and recurrent angina is discovered. Conclusions: We established a new mouse model of recurrent ischemia for mimicking recurrent angina, and find repeated ischemia induce trained immunity in macrophages.

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Key Words: Cardiac surgery; Cardiovascular disease; Ischemia reperfusion; Immunology; Inflammation