Physiol Biochem 2018;49:65-77 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000492840 DOI: 10.1159/000492840 © 2018 The Author(s) online:2222August, August, 2018 www.karger.com/cpb Published online: 2018 Published by S. Karger AG, Basel and Biochemistry Published www.karger.com/cpb Lugenbiel et al.: HDAC-Dependent Electrical Remodeling Accepted: 13 August, 2018
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Original Paper
Inhibition of Histone Deacetylases Induces K+ Channel Remodeling and Action Potential Prolongation in HL-1 Atrial Cardiomyocytes Patrick Lugenbiela,b Katharina Govorova,b Ann-Kathrin Rahma,b Teresa Wiedera,b Dominik Gramlicha,b Pascal Syrena,b Nadine Weiberga Claudia Seylera,c Hugo A. Katusa,b,c Dierk Thomasa,b,c a Department of Cardiology, University Hospital Heidelberg, Heidelberg, bHCR (Heidelberg Center for Heart Rhythm Disorders), Heidelberg, cDZHK (German Centre for Cardiovascular Research), Partner Site Heidelberg/Mannheim, University of Heidelberg, Heidelberg, Germany
Key Words Atrial fibrillation • Cardiac action potential • Epigenetic regulation • Histone deacetylase • Ion channel • Potassium channel • Tachypacing • Trichostatin A Abstract Background/Aims: Cardiac arrhythmias are triggered by environmental stimuli that may modulate expression of cardiac ion channels. Underlying epigenetic regulation of cardiac electrophysiology remains incompletely understood. Histone deacetylases (HDACs) control gene expression and cardiac integrity. We hypothesized that class I/II HDACs transcriptionally regulate ion channel expression and determine action potential duration (APD) in cardiac myocytes. Methods: Global class I/II HDAC inhibition was achieved by administration of trichostatin A (TSA). HDAC-mediated effects on K+ channel expression and electrophysiological function were evaluated in murine atrial cardiomyocytes (HL-1 cells) using real-time PCR, Western blot, and patch clamp analyses. Electrical tachypacing was employed to recapitulate arrhythmia-related effects on ion channel remodeling in the absence and presence of HDAC inhibition. Results: Global HDAC inhibition increased histone acetylation and prolonged APD90 in atrial cardiomyocytes compared to untreated control cells. Transcript levels of voltage-gated or inwardly rectifying K+ channels Kcnq1, Kcnj3 and Kcnj5 were significantly reduced, whereas Kcnk2, Kcnj2 and Kcnd3 mRNAs were upregulated. Ion channel remodeling was similarly observed at protein level. Short-term tachypacing did not induce significant transcriptional K+ channel remodeling. Conclusion: The present findings link class I/II HDAC activity to regulation of ion channel expression and action potential duration in atrial cardiomyocytes. Clinical implications for HDAC-based antiarrhythmic therapy and cardiac safety of HDAC inhibitors require further investigation. © 2018 The Author(s) Published by S. Karger AG, Basel
Dierk Thomas, MD
Department of Cardiology, Medical University Hospital Heidelberg Im Neuenheimer Feld 410, D-69120 Heidelberg (Germany) Tel. +49 6221 568855, Fax +49 6221 565514, E-Mail
[email protected]
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Physiol Biochem 2018;49:65-77 Cellular Physiology Cell © 2018 The Author(s). Published by S. Karger AG, Basel DOI: 10.1159/000492840 and Biochemistry Published online: 22 August, 2018 www.karger.com/cpb
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Lugenbiel et al.: HDAC-Dependent Electrical Remodeling
Introduction
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Electrophysiological properties of cardiomyocytes are governed by ion channels, pumps and exchangers. Ion channel dysfunction represents a key factor in the pathophysiology of heart rhythm disorders [1-5]. Specifically, alterations of ion channel expression contribute to the initiation and maintenance of cardiac arrhythmias [6, 7]. Current pharmacological antiarrhythmic therapy is limited by reduced effectiveness and side effects in a significant subgroup of patients [8-10]. Targeted therapeutic approaches based on underlying molecular mechanisms may improve future antiarrhythmic management. However, specific mechanisms contributing to defective regulation of ion channel expression during cardiac arrhythmia are poorly understood. We hypothesized that regulation of gene expression programs through epigenetic PA52CH15-McKinsey 4 December 2011 9:30 modulation may affect cardiac electrophysiology andARI promote arrhythmogenesis. DNA within the nucleus of eukaryotic cells is packaged Figure 1 together with histones and non-histone proteins in A a dynamic manner as chromatin [11]. Histones are Trichostatin A (TSA) subject to modification, including acetylation and deacetylation mediated by histone acetyltransferases a Surface Zinc (HATs) and histone deacetylases (HDACs), recognition Linker binding Surface Linker Zinc respectively [12]. Deacetylation of histone lysine recognition region binding residues results in chromatin condensation and O O modification of gene transcription. In addition, OH TSA HDACs interact with non-histone proteins such as NH H3C CH3 CH3 transcription factors, co-activators and co-repressors N [13]. Recent reports revealed that HDACs are CH3 A B involved in cardiac8 hypertrophy [14-16], fibrosis [17O 20 19] and metabolism [20]. Furthermore, QT-interval Control MS-275 N ** 6 TSA NH prolongation and ventricular * *** arrhythmias have been 15 B ControlO TSA NH NH2 observed during small molecule HDAC inhibition for 4 ** 17 kD 10 Ac-H3 anticancer therapy in humans, suggesting cardiac O 2 Butyrate 37 kD GAPDHO electrophysiological effects of HDAC inhibition [215 * O * 24]. 20 OH Control CH3 ** ** 0 0 Romidepsin CH3 The mechanisms underlying electrophysiological TSA * HN 15 HN actions of HDAC modulation are not known. This H3C S O O OH study was designed to investigate transcriptional and Valproic acid H3C 10 S HN CH3 functional effects of HDAC inhibition on repolarizing H3C 5 NH O Ctrl. TSA this end, the hydroxamic cardiac K+ channels. To C CH3 H3C 61 kD KCNQ1 O O 0 37 kD acid trichostatin A (TSA), 3 an established and GAPDH O 55 kD highly specific small molecule HDAC inhibitor with TREK-1 Fig. 1. Increased37 histone acetylation after kD 2 GAPDH * antitumor activity [25-28], was employed. At the pan-HDAC inhibition by trichostatin A 43 kD KCNJ2 37 kDstructure (C H N O ) molecular level, TSA serves as pan-HDAC inhibitor of bGAPDH (TSA). A. Chemical Efficacy of HDACi in preclinical models17of heart 22 2 failure 3 1 48 kD class I (HDACs 1, 2, 3, and 8) and II HDACs* (HDACs KCNJ3 of TSA. Functional domains of the 37 kD GAPDH ** 4, 5, 6, 7, 9, and 10) at nanomolar concentrations Scriptaid Apicidin-D 48 kD SAHAB. Protein levels compound are indicated. KCNJ5 0 (pan-HDACi) (class I HDACi) 37 kD(pan-HDACi) by reversibly binding to the catalytic site of the GAPDH of acetylated histone H3 (Ac-H3) were 150 kD HCN4 Mouse Rat Mouse enzyme [29]. TSA is a zinc-dependent HDAC inhibitor GAPDH 37 kD analyzed by Western blot in HL-1 cellsTAC TAC and MI DOCA 85 kD that harbors three distinct domains (Fig. 1 A) [30]. KCND3 incubated with 100 37 kD nM TSA for 12 hours, GAPDH First, the surface recognition site is formed by an followed by quantification of optical TSA Valproic acid aromatic group and interacts with the binding pocket density normalized to (weak glyceraldehyde (pan-HDACi) class I HDACi) of HDAC molecules. Second, a zinc binding domain 3-phosphate dehydrogenase (GAPDH). Rat Mouse 2+ coordinates to the active site of Zn ion. Third, the Immunoblots (upper and TAC, MI, AngII,mean SHR TAC, MI, AngII, ISO, panel) linker region connects the surface recognition site values (±SEM) Hop-Tg, Beclin-Tg Mouse TSA obtained following TAC to the zinc binding domain. Specifically, structural application (n=3) or control treatment studies using TSA bound to HDAC revealed that the (n=3) (lower panel) are shown; *p
