AAV-mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure

Basic Research in Cardiology (2021) 116:38 https://doi.org/10.1007/s00395-021-00880-w ORIGINAL CONTRIBUTION AAV‑mediated expression of NFAT decoy oligonucleotides protects from cardiac hypertrophy and heart failure Anca Remes1,3,4 · Andreas H. Wagner3 · Nesrin Schmiedel1,4 · Markus Heckmann2 · Theresa Ruf1,2 · Lin Ding1,4 · Andreas Jungmann2 · Frauke Senger1,4 · Hugo A. Katus2 · Nina D. Ullrich3 · Norbert Frey1,2,4 · Markus Hecker3 · Oliver J. Müller1,4 Received: 4 August 2020 / Accepted: 18 May 2021 © The Author(s) 2021 Abstract Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in hypertension-induced myocardial hypertrophy ultimately leading to heart failure. Here, we aimed at neutralizing four members of the NFAT family of transcription factors as a therapeutic strategy for myocardial hypertrophy transiting to heart failure through AAV-mediated cardiac expression of a RNA-based decoy oligonucleotide (dON) targeting NFATc1-c4. AAV-mediated dON expression markedly decreased endothelin-1 induced cardiomyocyte hypertrophy in vitro and resulted in efficient expression of these dONs in the heart of adult mice as evidenced by fluorescent in situ hybridization. Cardiomyocyte-specific dON expression both before and after induction of transverse aortic constriction protected mice from development of cardiac hypertrophy, cardiac remodeling, and heart failure. Singular systemic administration of AAVs enabling a cell-specific expression of dONs for selective neutralization of a given transcription factor may thus represent a novel and powerful therapeutic approach. Keywords Heart failure · Cardiac hypertrophy · Transcription factor · NFAT · Decoy oligonucleotide · Adeno-associated virus Introduction Although treatment options have significantly improved prognosis and quality of life of patients diagnosed with heart failure, current therapies at best offer reduced disease progression but not a real cure [30]. Cardiac hypertrophy is initiated by various stress signals, such as growth factors Anca Remes, Andreas H. Wagner, Markus Hecker and Oliver J. Müller contributed equally to this work. * Oliver J. Müller 1 Department of Internal Medicine III, University Hospital Schleswig-Holstein and University of Kiel , Arnold‑Heller‑Str. 3 , Kiel, Germany 2 Internal Medicine III, University Hospital Heidelberg, Heidelberg, Germany 3 Institute of Physiology and Pathophysiology, Heidelberg University, Heidelberg, Germany 4 German Centre for Cardiovascular Research , Partner Site Hamburg/Kiel/Lübeck , Kiel, Germany and pressure or volume overload [12]. Although this state is compensatory by temporarily preserving cardiac output, sustained pathological myocardial hypertrophy is associated with increased risk of heart failure, arrhythmias and sudden death [12, 42]. Myocardial hypertrophic growth is a highly intricate process governed by activation of the calcineurin-NFAT signaling pathway [32, 45]. Following dephosphorylation by calcineurin, transcriptional activation of the NFAT family members c1 to c4 regulates central processes leading to heart failure, such as myocyte growth, extracellular matrix deposition and re-activation of the fetal gene program [17, 41]. A number of studies have delineated the major importance of NFAT in the development of cardiac hypertrophy. Cardiac-specific activation of the calcineurin-NFAT pathway was sufficient to induce myocardial hypertrophy [33], whereas both pharmacological inhibition [24] and genetic deletion of NFATc2 and c3 [2, 46] alleviated pathological remodeling in animal models. Therefore, reducing the transcriptional activity of NFAT could represent a valuable treatment option for cardiac hypertrophy and heart failure. 13 Vol.:(0123456789) 38 Page 2 of 12 One approach of interfering with abnormal activity of transcription factors for therapeutic purposes is represented by decoy oligodeoxynucleotides (dODNs). These are short (10–25 bp) sequences of double-stranded DNA mimicking the consensus binding site of the target transcription factor, which upon cellular entry specifically neutralize it and hence inhibit transcription of its target genes [15]. These nucleic acid-based drugs have been proven to be safe and effective in various preclinical and clinical trials focusing on diverse disease models [29]. Until now, the most frequent route of administration employed is local delivery, which requires sustained application of the active dODNs [8, 9]. In terms of future therapy and depending on the target organ, this approach is not always feasible. Here, we show that single injection of an adeno-associated viral vector (AAV9) is sufficient to express a decoy hairpin RNA oligonucleotide (dON) neutralizing NFATc1c4 in cardiomyocytes in vivo. This singular treatment before or after subjecting mice to transverse aortic constriction (TAC) was associated with a dramatic improvement in cardiac function and reduced remodeling, and therefore broaden the paradigm of decoy oligo(deoxy)nucleotides as nucleic acid-based therapeutics. Materials and methods Primary cardiomyocytes isolation and culture Primary neonatal rat cardiomyocytes (NRVCMs) were isolated according to previously established protocols [22]. Experiments were executed under the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes with approval of the local authorities in Kiel (permission number 1085). In brief, left ventricles from 2-day-old Wistar rats were harvested and digested in the presence of 0.6 mg/mL pancreatin (Sigma-Aldrich, Munich, Germany) and 0.5 mg/ mL collagenase II (Worthington Biochemical Corporation, Lakewood, USA) at 37 °C. The resulting cell suspension was filtered through a cell strainer and afterwards centrifugation on a Percoll gradient (GE Healthcare, Chicago, USA) was performed to ensure separation from fibroblasts. Next, NRVCMs were cultured in complete DMEM medium containing 10% fetal bovine serum, 2 mmol/L L-glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Thermo Fischer Scientific, Darmstadt, Germany). DNA dODN technology DNA-based dODNs were designed to form a hairpin structure by intramolecular hydrogen bond formation and to contain the promoter binding site of NFAT1-4 (Biomers, 13 Basic Research in Cardiology (2021) 116:38 Ulm, Germany). A 5′-Atto-590 labeled dODN was used for assessment of cellular uptake efficiency. The sequences of the dODNs used in our study are as follows: hpNFAT cons dODN: 5′-GAGTGGAAACATACAGCCACTGAA ACAGTGGCTGTATGTTTCCACTC-3′ and hpNFAT mut dODN: 5′-GAGCTTAAACATACAGCCACTGAAACA GTGGCTGTATGTTTCCACTC-3′. Decoys were dissolved in sterile TEN buffer to a concentration of 500 μmol/L, incubated at 95 °C for 5 min, followed by gradual cooling down, to induce hybridization of complementary base pairs and hairpin structure formation. Successful hybridization was proven by agarose gel electrophoresis. AA (...truncated)