Introduction: Heart failure represents the leading cause of death in the Western world. However, recent treatment options focus solely on reducing the severity of symptoms. Previous studies have underlined the substantial role of nuclear factor of activated T cells (NFAT) in the regulation of cardiac growth, fetal gene expression and extracellular matrix deposition. Collectively, these processes contribute to pathological myocardial hypertrophy and ultimately lead to heart failure.

Purpose: Our aim is to modulate the activity of NFAT 1-4 as a potential therapeutic strategy for heart failure using decoy oligonucleotides delivered into cardiomyocytes by adeno-associated virus (AAV) vectors.

Methods: NFAT consensus binding sequence (hpNFAT dON, 40 bp) and mutated control oligonucleotide were cloned into an AAV plasmid under the control of H1 promotor.  In vitro experiments were performed in neonatal rat ventricular cardiomyocytes (NRVCMs) using AAV6 vectors. Endothelin-1 (ET-1) was used as a pro-hypertrophic stimulus, and mRNA levels of fetal genes were measured by real-time PCR. Cell size was assessed by a-actinin immunocytochemistry. For in vivo studies, AAV9 vectors expressing hpNFAT dON were injected 2 days after induction of cardiac hypertrophy by transverse aortic constriction (TAC). Expression of dONs following transduction was demonstrated by fluorescent in situ hybridization (FISH). Cardiac function (ejection fraction, EF; fractional shortening, FS) and left ventricular mass were measured by echocardiography. Myocardial fibrosis was assessed in frozen sections by Masson’s Trichrome staining.

Results: Transduction with AAV6 vectors expressing hpNFAT dONs markedly decreased ET-1 induced NRVCM hypertrophy, evidenced by a marked reduction in fetal gene programme and decrease of cardiomyocyte size as compared to controls. Tail-vein injection of the designed AAV9 into C57BL/6J mice led to efficient expression of the nucleic acid compound, evidenced by FISH. Importantly, in vivo experiments showed that although cardiac function severely deteriorated 6 weeks after TAC in control mice (EF: 58±6,4% in sham operated mice, 42±4% in TAC control mice), AAV9-mediated delivery of dONs after TAC resulted in preservation of cardiac function to basal levels (EF: 59±3,58%). Moreover, we could show that our treatment largely ameliorated cardiac hypertrophy, demonstrated by a reduction in HW/TL (26±2% decrease) and LV mass (41±2,3% decrease) as compared to controls. Additionally, reducing NFAT transcriptional activity led to marked reduction in extracellular matrix development in the myocardium.

Conclusion: Our study proves that continuous generation of dONs targeting NFAT transcriptional activity in cardiomyocytes potently inhibits cardiac hypertrophy caused by hemodynamic stress and improves left ventricular function. Antagonizing increased NFAT activity thus seems to be a promising approach for translation into treatment of heart failure.