Pharmacological inhibition of GRK5 improves cardiac function in experimental Takotsubo syndrome

K. Berger (69120 Heidelberg, Germany)¹, M. Dewenter (Heidelberg)², B. Bruns (Heidelberg)³, S. Nazir (Heidelberg)⁴, M. Baumann (Dortmund)⁵, J. Engel (Dortmund)⁶, F. Ballaschk (Dortmund)⁶, B. Klebl (Dortmund)⁶, J. Backs (Heidelberg)^4
1Heidelberg University, Medical Faculty Heidelberg, Institute of Experimental Cardiology, 69120 Heidelberg, Germany; Heidelberg University Hospital, Department of Internal Medicine VIII, 69120 Heidelberg, Germany; German Center for Cardiovascular Research (DZHK), Partner Site Heidelberg/Mannheim 69120 Heidelberg, Germany, Deutschland; ²Universitätsklinikum Heidelberg Molekulare Kardiologie und Epigenetik Heidelberg, Deutschland; ³Universitätsklinikum Heidelberg Klinik für Innere Med. III, Kardiologie, Angiologie u. Pneumologie Heidelberg, Deutschland; ⁴Universitätsklinikum Heidelberg Institut für experimentelle Kardiologie Heidelberg, Deutschland; ⁵Lead Discovery Center GmbH (LDC) Dortmund, Deutschland; ⁶Lead Discovery Center GmbH Dortmund, Deutschland

Background:
Takotsubo syndrome (TTS) is an acute, mostly transient form of heart failure. Although the condition gains recognition in research and clinical practice, underlying molecular mechanisms are poorly understood and targeted therapies largely missing. Genetic analyses performed on blood samples from TTS patients show a significant higher frequency of G protein-coupled receptor kinase 5 (GRK5) Leu41 compared to control groups, a mutation that is correlated with an enhanced function of GRK5. Yet it is unknown whether increased GRK5 activity plays a causative role in TTS and what molecular downstream mechanisms might be involved.

Aim: We aim to specify the role of GRK5 in the development of TTS and determine whether pharmacological inhibition of GRK5 can serve as a new therapeutic approach.

Methods and Results:
To model TTS in a preclinical vivo setting we used a mouse model of acute catecholamine stress that recapitulates multiple features of human TTS, such as transient contractile failure and acute myocardial damage. To evaluate the functional relevance of GRK5 activity in experimental TTS, we applied increasing dosages of novel lead compounds that inhibit GRK5 (GRK5i) 30 minutes prior to exposing mice to TTS via epinephrine injection. Echocardiographic analysis revealed a dose-dependent rescue of cardiac ejection fraction (EF) 1 hour after epinephrine injection (EF: 19,2% (0mg/kg BW) vs. 41,6% (1mg/kg BW) vs. 46,8% (10mg/km BW) vs. 69,0% (100mg/kg BW), n=5-6/group, p<.0001). In a follow-up study on long-term effects, cardiac function between 30 minutes to 48 hours after epinephrine injection benefitted significantly from preventive GRK5i treatment. EF in the inhibition group was completely restored 24 hours after epinephrine injection, whereas EF in the vehicle group recovered not until day five. Finally, applying GRK5i 30 minutes after epinephrine injection also mitigated contractile dysfunction within the first 48 hours, showing that GRK5 inhibition has not only beneficial effects in a preventive, but also in a therapeutic approach.

Besides its role in regulating beta Adrenoceptor desensitization GRK5 acts as class IIa histone deacetylase (HDAC) kinase, thereby regulating transcription. Time course analysis of cardiac tissue from mice with TTS revealed that class IIa HDAC phosphorylation increased already 15 minutes after epinephrine injection, peaking at 1 hour and slowly decreasing afterwards. mRNA levels of NR4A1, a previously characterized critical mediator of acute cardiac fatigue, also reached a maximum after 1 hour. Activation patterns correlated with observed contractile dysfunction in TTS, indicating an involvement of early transcriptional activation in development of TTS. In vitro experiments performed in neonatal cardiomyocytes and COS cells confirmed potent effects of the GRK5i on class IIa HDAC phosphorylation and HDAC localization. In vivo, we also observed a decrease in HDAC phosphorylation after administering GRK5i, implying that modulation of acute maladaptive gene transcription is indeed underlying the functional rescue upon GRK5i treatment.

Conclusion:
Our preclinical in vivo studies suggest that GRK5 inhibition is a promising pharmacological approach to rescue cardiac function in TTS. We identified the inhibition of a signaling axis involving GRK5-dependent HDAC phosphorylation and subsequent acute maladaptive gene expression as a potential downstream mechanism of the GRK5i benefit in TTS.