Unravelling the signaling pathway downstream of the neurokinin receptor in atrium: discovering novel anti-arrhythmic targets for treatment of atrial fibrillation

Abstract

Abstract Background Stimulation of neurokinin receptor (NKR) 3 is anti-fibrillatory by prolonging the action potential duration (APD) of atrial cardiomyocytes via inhibition of a background potassium current (1). The NKR is a Gq/11 protein-coupled receptor, but the downstream intracellular pathway and the end-effector potassium (K+)-channel in atrium are unknown. We aimed at unravelling the components of this pathway in atrial cardiomyocytes. Methods Rabbit (New Zealand White) atrial cardiomyocytes were isolated from Langendorff-perfused hearts by enzymatic dissociation. Human immortalized atrial myocytes (HiAMs) were developed, and kindly provided by our collaborators (2). Adult human atrial cardiomyocytes were obtained by enzymatic dissociation from left atrial appendages (LAA) routinely removed from atrial fibrillation (AF) patients undergoing minimally invasive surgical pulmonary vein isolation in the clinics. Action potentials (APs) and K+ currents were recorded at 36.5°C with the amphotericin-B-perforated patch clamp technique. RNA was isolated from rabbit atrial tissue and sequenced on an Illumina HiSeq4000 platform. Results In rabbit atrial cardiomyocytes, stimulation of NKR3 prolonged APD by ∼50% and inhibited an background outward K+-current by ∼45%. Inhibition of phosphokinase C (PKC) did not prevent APD prolongation by NKR3 stimulation, but application of a diacylglycerol (DAG) analog inhibited an outward K+-current by ∼40% indicating that DAG, but not PKC, is downstream of NKR3. Inhibition of phosphatidylcholine-specific phospholipase C (PC-PLC), but not of phosphoinositide-specific PLC (PI-PLC), reduced APD increase by NKR3 stimulation by five-fold, indicating PC-PLC as the intracellular signaling molecule. In HiAMs, direct PLC activation prolonged APD by 34%. RNA sequencing analysis revealed potential end-targets of the NKR3 pathway in rabbit. Inhibition of small conductance potassium (KCNN2/KCNN3) or TASK-1 (KCNK3) channels (prolonging APD by ∼15%) did not abolish the APD prolongation effect by subsequent NKR3 stimulation (∼50%), indicating that these channels are not the end-effector K+-channels inhibited by NKR3 stimulation. Inhibition of constitutive KACh channels had little effect on APD at baseline. Nevertheless, APD shortening by agonist-induced KACh channel activity could be completely reversed by NKR3 stimulation, indicating that increased outward KACh current can be fully counterbalanced by the reduction in K+ background current. In human atrial cardiomyocytes from AF patients, stimulation of NKR3 prolonged APD by 25%, Conclusions PC-PLC and DAG, but not PI-PLC and PKC, are intracellularly downstream of NKR3 in atrial rabbit cardiomyocytes. Stimulation of the NKR pathway may have an anti-arrhythmic potential in patients with AF.