Cardiovascular Pharmacology

Arrhythmia mechanisms in human induced pluripotent stem cell-derived cardiomyocytes Despite major efforts by clinicians and researchers, cardiac arrhythmia remains a leading cause of morbidity and mortality in the world. Experimental work has relied on combining high-throughput strategies with standard molecular and electrophysiological studies, which are, to a great extent, based on the use of animal models. As this poses major challenges for translation, the progress in the development of novel antiarrhythmic agents and clinical care has been mostly disappointing. Recently, the advent of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) has opened new avenues for both basic cardiac research and drug discovery: now there is an unlimited source of CMs of human origin, both from healthy individuals and patients with cardiac diseases. Understanding arrhythmic mechanisms is one the main use-cases of hiPSC-CMs, in addition to pharmacological cardiotoxicity and efficacy testing, in vitro disease modeling, developing patient-specific models and personalized drugs, and regenerative medicine. Here, we review the advances that the hiPSC-based modeling systems have brought so far regarding the understanding of both arrhythmogenic triggers and substrates, while also briefly speculating about the possibilities in the future. Corresponding author: Jussi T. Koivumäki, Arvo Ylpön katu 34, FI-33520 Tampere, Finland, +358 40 5813 290, jussi.koivumaki@iki.fi This is an open access article distributed under the Creative Commons Attribution License 4.0 (CCBY), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

A Perspective on Personalized Therapies in Hypertrophic Cardiomyopathy A dominant mechanism of sudden cardiac death in the young is the progression of maladaptive responses to genes encoding proteins linked to hypertrophic cardiomyopathy (HCM). Most are mutant sarcomere proteins that trigger the progression by imposing a biophysical defect on the dynamics and levels of myofilament tension generation. We discuss approaches for personalized treatments that are indicated by recent advanced understanding of the progression Corresponding Author: R. John Solaro, PhD, Department of Physiology and Biophysics (M/C 901), University of Illinois at Chicago, College of Medicine, 835 South Wolcott Avenue, Chicago, IL 60612, eMail: solarorj@uic.edu Online date: 00 00, 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Heat shock protein 70 is associated with cardioversion outcome and recurrence of symptomatic recent onset atrial fibrillation in hypertensive patients Accumulating evidence indicates that heat shock proteins (HSPs) may represent a suitable biomarker to predict atrial fibrillation (AF). We investigated the relation of circulating heat shock protein 70 (sHSP70) with inflammatory cytokines and recurrence of symptomatic recent onset AF (ROAF). We enrolled 90 patients with ROAF (duration from onset of symptoms ≤24 hours) and 30 controls. Patients received amiodarone for cardioversion and rhythm control. The association of serum HSP70, interleukin-2 (sIL-2) and interleukin-4 (sIL-4) with the presence, cardioversion and AF recurrence within a year was investigated. TLR4 signaling dependence for IL-2 and IL-4 induction in response to stimulation with HSP70 was tested in rat aortic vascular smooth muscle cell (SMC) cultures. Patients had higher sHSP70 and sIL-2 and lower sIL-4 compared to controls. sHSP70 was independently associated with ROAF (P=0.005) and correlated with sIL-2 (r =0.494, P<0.001) and sIL-4 (r=-0.550, P<0.001). By 48 hours 71/90 patients were cardioverted, with non-cardioverted patients having higher sHSP70 and sIL-2 and lower sIL-4, which were the only independent factors associated with cardioversion. AF recurred in 38/71 cardioverted patients in one year. A cut-off value of sHSP70 ≥0.65 ng/ml and sIL-2 ≥0.21 pg/ml were the only independent factors associated with AF recurrence (HR: 3.311, 95% CI: 1.503-7.293, P=0.003 and HR: 3.144, 95% CI: 1.341-7.374, P=0.008, respectively). Exposure of SMC to HSP70 in vitro increased the expression of IL-2 (5x) and IL-4 (1.5x) through TLR4-dependent and receptor-independent mechanisms. In conclusion, sHSP70 and sIL-2 might constitute a prognostic tool for determining the cardioversion and recurrence likelihood in ROAF. Corresponding author: Dr. Angelos G. Rigopoulos, MD, FESC, Mid-German Heart Center, Department of Internal Medicine III, Division of Cardiology, Angiology and Intensive Medical Care, University Hospital Halle, Martin-Luther-University Halle-Wittenberg, Ernst-Grube-Strasse 40, D-06120 Halle (Saale), Germany, Tel.: +49 176 79844001, Fax: +49 345 5572072 E-Mail: angelos.rigopoulos@gmail.com Online date: 00 00, 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Circular RNA circ-BANP regulates ox-LDL-induced endothelial cell injury through targeting the miR-370/TXNIP axis Dysfunction of endothelial cells is now recognized as an important contributor to the pathogenesis of atherosclerosis (AS). Circular RNAs (circRNAs) have been demonstrated to be involved in AS pathogenesis. The purpose of this study was to explore the biological action of circRNA BTG3 associated nuclear protein (circ-BANP, hsa_circ_0040824) on the dysfunction of human umbilical vein endothelial cells (HUVECs) induced by oxidized low-density lipoprotein (ox-LDL). The levels of circ-BANP, miR-370 and thioredoxin-interacting protein (TXNIP) were gauged by quantitative real-time polymerase chain reaction (qRT-PCR) or western blot. Subcellular fractionation assay was used to determine the localization of circ-BANP, and ribonuclease R (RNase R) assay was performed to evaluate the stability of circ-BANP. Cell viability, apoptosis, migration, invasion and tube formation abilities were assessed by the Cell Counting Kit-8 (CCK-8), flow cytometry, transwell, and tube formation assays. The levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). Targeted relationships among circ-BANP, miR-370 and TXNIP were confirmed by a dual-luciferase reporter assay. Our data showed that circ-BANP expression was up-regulated in AS blood and ox-LDL-induced HUVECs. The inhibition of circ-BANP promoted cell viability, migration, invasion, tube formation, and repressed cell inflammation and apoptosis in ox-LDL-induced HUVECs, demonstrating that circ-BANP silencing alleviated ox-LDL-induced HUVEC injury. Mechanistically, circ-BANP directly targeted miR-370. Moreover, miR-370 mediated the regulation of circ-BANP in ox-LDL-induced cell injury in HUVECs. TXNIP was a target of miR-370, and miR-370 overexpression relieved ox-LDL-induced HUVEC injury by down-regulating TXNIP. Furthermore, circ-BANP modulated TXNIP expression by targeting miR-370. Our findings demonstrated that circ-BANP regulated ox-LDL-induced cell injury in HUVECs at least in part through targeting the miR-370/TXNIP axis, illuminating circ-BANP as a potential target for AS detection and treatment. Corresponding author: Lijun Gao, Department of General Internal Medicine, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, Zhongjing Building, No. 16369, Jingshi Road, Lixia District, Jinan, Shandong, 250014, China, Tel: +86-0531-68617110; E-mail: gljsdzhyy0812@163.com Online date: 00 00, 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Evaluation of sacubitril/valsartan initiation in outpatient heart failure patients Despite sacubitril/valsartan being on the market since 2015, clinicians are still determining the best way to initiate therapy in order to optimize outcomes and minimize potential for side effects. The purpose of this study is to investigate real-world outpatient experience of prescribing sacubitril/valsartan therapy based on appropriate patient selection, dosing conversion, and tolerability. This retrospective cohort study evaluated patients prescribed sacubitril/valsartan therapy in cardiology clinics associated with an academic institution between February 1, 2016 and August 30, 2018. Patients were excluded if they were less than 18 years of age, enrolled in a clinical trial involving sacubitril/valsartan, or had insufficient data. The primary outcome was to determine how many heart failure patients initiated on sacubitril/valsartan were done so appropriately based on guideline and package insert recommendations. Select secondary outcomes included rates of adverse events and need for adjustment of concomitant heart failure medications. A total of 250 patients were included in this study. For the primary outcome, 125 patients (50%) were appropriately initiated on sacubitril/valsartan. Those who were inappropriately initiated on the medication experienced more symptoms of hypotension (16% in appropriate start group vs. 28% in inappropriate start group; p=0.022) and required more dose decreases of sacubitril/valsartan (6% in appropriate start group vs. 13% in inappropriate start group; p=0.049). In outpatient clinical practice, almost half of patients initiated on sacubitril/valsartan were done so outside of guideline recommendations, which was associated with an increased risk of hypotension and dose reductions. Corresponding Author: Lindsey Rerick, PharmD, Allegheny Health Network, West Mifflin, PA UNITED STATES Online date: 00 00, 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Lipoprotein(a) reduction with proprotein convertase subtilisin/kexin type 9 inhibitors: a systematic review and meta-analysis. Lipoprotein(a) (Lp(a)) is a cardiovascular factor, for which there is no approved specific lowering treatment. Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors have been shown to have lowering effects on Lp(a). Aim of this systematic review is to synthesize the current literature and quantify the effects of PCSK9 inhibitors on the serum Lp(a) levels in human subjects. Double-blind, phase 2 or 3, randomized controlled trials comparing PCSK9 inhibitors (alirocumab or evolocumab) to placebo and/or ezetimibe and/or other lipid lowering therapy were deemed eligible for inclusion. We searched MEDLINE (via PubMed), CENTRAL, Scopus and Web of Science as of 17 June 2020. Quality assessment was performed using the Revised Cochrane risk-of-bias tool for randomized trials (RoB2). Forty-three studies were identified (64107 patients randomized) and 41 studies were included in the quantitative analysis. PCSK9 inhibitors reduced Lp(a) levels by -26.7% (95% CI -29.5% to -23.9%) with a significant heterogeneity within studies. There was significant difference in Lp(a) change from baseline according to comparator (placebo: mean -27.9%, 95% CI -31.1% to -24.6% vs. ezetimibe: mean -22.2%, 95% CI -27.2% to -17.2%, p=0.04) and duration of treatment (≤12 weeks: mean -30.9%, 95% CI -34.7% to -27.1% vs. >12 weeks: mean -21.9%, 95% CI -25.2% to -18.6%, p<0.01). Meta-regression analysis showed that only the mean percentage change from baseline LDL-C due to the intervention is significantly associated with the effect size difference (p<0.0001). PCSK9 inhibitors reduced LDL-C by -54% (95% CI -57.6% to -50.6%). There is substantial efficacy of the currently approved PCSK9 inhibitors in the lowering of Lp(a) levels. Dedicated RCTs are needed to establish the benefit of this intervention. Corresponding author: George Giannakoulas, MD, PhD, Associate Professor of Cardiology, Aristotle University of Thessaloniki, AHEPA Hospital, Cardiology Department, Stilp. Kiriakidi 1, 54637, Thessaloniki, Greece, Tel: +302313303589, Email: ggiannakoulas@auth.gr Online date: 00 00, 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Is spironolactone the preferred renin-angiotensin-aldosterone inhibitor for protection against COVID-19? The high mortality of specific groups from COVID-19 highlights the importance of host-viral interactions and the potential benefits from enhancing host defenses. SARS-CoV-2 requires angiotensin converting enzyme (ACE)2 as a receptor for cell entry and infection. While both ACE inhibitors and spironolactone can upregulate tissue ACE2, there are important points of discrimination between these approaches. The virus requires proteolytic processing of its spike protein by transmembrane protease receptor serine type 2 (TMPRSS2) to enable binding to cellular ACE2. Since TMPRSS2 contains an androgen promoter, it may be downregulated by the antiandrogenic actions of spironolactone. Furin and plasmin also process the spike protein. They are inhibited by protease nexin 1 or serine E2 (PN1) that is upregulated by angiotensin II but downregulated by aldosterone. Therefore, spironolactone should selectively downregulate furin and plasmin. Furin also promotes pulmonary edema while plasmin promotes hemovascular dysfunction. Thus, a downregulation of furin and plasmin by PN1 could be a further benefit of MRAs beyond their well established organ protection. We review the evidence that spironolactone may be the preferred RASSi to increase PN1 and decrease TMPRSS2, furin and plasmin activities and thereby to reduce viral cell binding, entry, infectivity and bad outcomes. This hypothesis requires direct investigation. Address for correspondence: Christopher Wilcox, MD, PhD, 3800 Reservoir Road, NW, PHC F6003, Washington, DC 20007, Fax: 877-625-1483, Email: wilcoxch@georgetown.edu Sources of Funding: Work in CSW's laboratory is supported by the Smith-Kogod Family Trust, the Gildenhorn-Speisman Family Trust, the Georgetown University Hypertension Research Center and the Walters Family Chair of Cardiovascular Research, all located in Washington DC. Disclosures: CSW, none. BP, none. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal. Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Intermittent optogenetic tachypacing of atrial engineered heart tissue induces only limited electrical remodelling Atrial tachypacing is an accepted model for atrial fibrillation (AF) in large animals and in cellular models. Human induced pluripotent stem cells derived cardiomyocytes (hiPSC-CM) provide a novel, human source to model cardiovascular diseases. Here we investigated whether optogenetic tachypacing of atrial-like hiPSC-CMs grown into engineered heart tissue (aEHT) can induce AF-remodeling. After differentiation of atrial-like cardiomyocytes from hiPSCs using retinoic acid, aEHTs were generated from ∼1 million atrial-like hiPSC-CMs per aEHT. AEHTs were transduced with lentivirus expressing channelrhodopsin-2 to enable optogenetic stimulation by blue light pulses. AEHTs underwent optical tachypacing at 5 Hz for 15 s twice a minute over three weeks and compared to transduced spontaneously beating isogenic aEHTs (1.95±0.07 Hz). Force and action potential duration did not differ between spontaneously beating and tachypaced aEHTs. Action potentials in tachypaced aEHTs showed higher upstroke velocity (138±15 V/s vs. 87±11 V/s, n=15-13/3; p=0.018), possibly corresponding to a tendency for more negative diastolic potentials (73.0±1.8 mV vs. 68.0±1.9 mV; p=0.07). Tachypaced aEHTs exhibited a more irregular spontaneous beating pattern (beat-to-beat scatter: 0.07±0.01 vs. 0.03±0.004 s, n=15-13/3; p=0.008). Targeted expression analysis showed higher RNA levels of KCNJ12 (Kir2.2, inward rectifier (IK1); 69±7 vs. 44±4, p=0.014) and NPPB (NT-proBNP; 39690±4834 vs. 23671±3691; p=0.024). Intermittent tachypacing in aEHTs induces some electrical alterations found in AF and induces an arrhythmic spontaneous beating pattern, but does not affect resting force. Further studies using longer, continuous, or more aggressive stimulation might clarify the contribution of different rate patterns on the changes in aEHT mimicking the remodeling process from paroxysmal to persistent atrial fibrillation. Address for Correspondence: PD. Dr. med. Torsten Christ, Institut für Experimentelle Pharmakologie und Toxikologie, Universitätsklinikum Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany, Tel +49 40 7410 52180, Fax+49 40 7410 54876, E-mail address: t.christ@uke.de * These authors contributed equally # Current Address: Nanion Technologies GmbH, Ganghoferstraße 70a, 80339 München, Germany. This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial License 4.0 (CCBY-NC) , where it is permissible to download, share, remix, transform, and buildup the work provided it is properly cited. The work cannot be used commercially without permission from the journal. Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Knockdown of long non-coding RNA SNHG14 protects H9c2 cells against hypoxia-induced injury by modulating miR-25-3p/KLF4 axis in vitro Cyanotic congenital heart disease (CCHD) is the main cause of death in infants worldwide. Long non-coding RNAs (lncRNAs) have been pointed to exert crucial roles in development of CHD. The current research is designed to illuminate the impact and potential mechanism of lncRNA SNHG14 in CCHD in vitro. The embryonic rat ventricular myocardial cells (H9c2 cells) were exposed to hypoxia to establish the model of CCHD in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to examine relative expressions of SNHG14, miR-25-3p and KLF4. Cell viability was determined by MTT assay. Lactate dehydrogenase (LDH) was measured by an LDH assay kit. Apoptosis-related proteins (Bax and Bcl-2) and KLF4 were detected by Western Blot. The targets of SNHG14 and miR-25-3p were verified by dual-luciferase reporter (DLR) assay. SNHG14 and KLF4 were up-regulated, while miR-25-3p was down-regulated in hypoxia-induced H9c2 cells and cardiac tissues of patients with CCHD compared with their controls. Knockdown of SNHG14 or overexpression of miR-25-3p facilitated cell viability, while depressing cell apoptosis and release of LDH in hypoxia-induced H9c2 cells. MiR-25-3p was a target of SNHG14 and inversely modulated by SNHG14. MiR-25-3p could directly target KLF4 and negatively regulate expression of KLF4. Repression of miR-25-3p or overexpression of KLF4 reversed the suppression impacts of sh-SNHG14 on cell apoptosis and release of LDH as well as the promotion impact of sh-SNHG14 on cell viability in hypoxia-induced H9c2 cells. Sh-SNHG14 protected H9c2 cells against hypoxia-induced injury by modulating miR-25-3p/KLF4 axis in vitro. Corresponding author: Zhaoyun Cheng, Address: Department of Cardiovascular Surgery, Fuwai Central China Cardiovascular Hospital, Heart Center of Henan Provincial People's Hospital, No.1, Fuwai Road, Zhengdong New District, Zhengzhou City, Henan Province, 451464, China. Phone number: 86-13903712068, Email: chengzhaoyun287@163.com Conflict of Interest: The authors have no conflict of interest to disclose. Received July 20, 2020 Accepted November 05, 2020 Online date: 00 00, 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

Leonurine Attenuates Myocardial Fibrosis through Upregulation of miR-29a-3p in Mice Post-myocardial Infarction Abstract: Myocardial fibrosis (MF) is a pathological process that accelerates cardiac remodeling in myocardial infarction (MI), and miR-29 has become one of the foci of research into MF. As an alkaloid extracted from Herba leonuri, leonurine (LE) has been found to be an effective natural active ingredient for inhibiting fibrosis in many preclinical experiments. However, whether LE protects against MF after MI through modifying miR-29 remains unclear. The present study aimed to investigate the therapeutic effects of LE on MF, and to elucidate the underlying mechanisms involved. A mouse model of MI was established, followed by administration of LE for 4 weeks. We found that LE effectively improved cardiac function, and attenuated fibrosis and cardiac remodeling in mice post-MI. In vitro, LE simultaneously inhibited proliferation and migration of neonatal mouse cardiac fibroblasts (CFs) exposed to angiotensin II (Ang II), and the activation of collagen synthesis and myofibroblast generation was markedly suppressed by LE. Notably, we found that all mature miR-29 family members were downregulated in the myocardial tissues of mice post-MI, while, LE significantly upregulated miR-29a-3p expression, and such upregulation was also detected in LE-treated CFs under Ang II stimulation. Knockdown of miR-29a-3p by a specific miRNA inhibitor upregulated the protein levels of TGF-β, Collagen III, and Collagen I in CFs, and completely reversed the antifibrotic effects of LE on CFs. Our study suggests that LE exerts cardioprotective effects against MF, possibly through the upregulation of miR-29a-3p. Corresponding author: Dr. Jiadan Yang. Department of Pharmacy, the First Affiliated Hospital of Chongqing Medical University, 1 Youyi Road, Yuanjiagang, Yuzhong District, Chongqing 400016, P.R. China. E-mail address: yangjiadan11@126.com The authors report no conflicts of interest. † These two authors contributed equally to this work. Online date: 00 00, 2020 Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.

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