DATA FILE: MODULATION OF BETA-ADRENOCEPTOR-STIMULATED cAMP SIGNALLING KINETICS BY GRK2 IN A CELLULAR MODEL

Abstract

In heart failure, increased levels of the G-protein coupled receptor kinase 2 (GRK2) lead to increased desensitization of beta-adrenoceptors and thus the heart is getting insensible to neurohormonal stimulation worsening the symptoms of heart failure. The presented study wants to elucidate the impact of GRK2 on beta-adrenoceptor stimulated cAMP signalling kinetics accountable for the heart’s contractility in a cellular model system. An EPAC-based cAMP FRET biosensor was used to measure intracellular cAMP levels in real-time. HEK293A cells stably expressing the biosensor and (I) endogenous levels of GRK2, (II) increased levels of GRK2, and (III) elevated levels of the dominant negative GRK2(K220R) mutant were established. Moreover, the GRK2 gene was knocked-out by the CRISPR/Cas9 system. Therefore, four lentiCRISPRv2 vectors were cloned coding for the Cas9 protein and oligos to guide it to specific sites in the GRK2 gene. After stable transfection and clone selection the protein content was analysed by SDS-PAGE, western blotting and immunodetection of GRK2. Also, the cutting site of the Cas9 protein was investigated by PCR amplification of the genomic DNA followed by Sanger sequencing and TIDE analysis to identify inserted base pairs and premature stop codons confirming the GRK2 knock-out. The sensor was fully characterized by fluorescence confocal microscopy and cAMP titration after cell permeabilization with digitonin in all cell lines. Beta-adrenoceptor cAMP signalling kinetics stimulated by increasing concentrations of isoproterenol was characterized by Emax and EC50. By stimulation of all cell lines with isoproterenol at Emax and pre-incubation with and without small molecule GRK2 inhibitor CMPD101 the effects of GRK2 and its inhibition on the beta-adrenoceptor stimulated cAMP signalling kinetics were elucidated. In addition to peak height and AUC, data was analysed by population kinetics using non-linear mixed effects modelling to also address the signalling curve shape and quantify kinetic rate constants. The presented cellular model can reproduce the cAMP diminishing effects of GRK2 in heart failure. Moreover, it reveals potential beneficial cAMP signalling enhancing effects of GRK2 inhibition by CMPD101. It can be of interest using this model to identify potential new GRK2 inhibitors and characterize them by their ability of elevating cAMP signalling kinetics. Moreover, the present model sets the foundation to further approximate to the in-vivo conditions by the translation to cardiomyocytes or human heart organoids or address the influence of GRK2 in different cellular compartments within more detail.