P4441Serum activity against specific G-protein coupled receptors is associated with the severity of orthostatic symptoms in patients with POTS
Authors: Kharraziha I , Medic Spahic J , Ricci F , Persson-Tholin J , Persson M , Firth K , Axelsson J , Sutton R , Fedorowski A , Hamrefors V 
Published: October 2019

Abstract

Background
Postural orthostatic tachycardia syndrome (POTS) is characterised by an excessive heart rate increase upon standing and orthostatic intolerance. Previous studies indicate autoimmune involvement mediated via G-protein coupled receptors (GPCRs) as a possible aetiology.

Purpose
To test if serum from POTS patients and controls activates selected GPCRs and if the activation associates with the severity of symptoms.

Methods
A total of 34 POTS patients (age 28.9 [9.9] years; 29 women) and a population-based control group of 25 healthy subjects (age 30.7 [8.6] years; 21 women) were included. The subjects performed an active standing test and completed the orthostatic hypotension questionnaire (OHQ) including ten items related to orthostatic intolerance. The OHQ composite score was calculated (range 0–10).
Sera were analysed by a FRET-based reporter system (Tango GeneBLAzer, Thermo Fischer) based on a beta-arrestin-linked transcription factor driving transgenic betalactamase transcription. HEK293M-cells over-expressing one of the GPCRs, adrenergic alpha-1 (ADRA1), adrenergic beta-2 (ADRB2), muscarinic type-2 (CHRM2) and opioid-receptor-like 1 (OPRL1) receptor were plated in 96-well optical plates and allowed to re-attach during 48 hrs. The cells were treated with 10% sera diluted in RPMI for 5 hours, followed by addition of the FRET-substrate, incubation for 60 min and quantification analysis in a CLARIOStar multi-purpose plate reader.
Quantification of the activation of the GPCRs was log-transformed and related to the OHQ composite and individual scores in age-adjusted linear regression models. OHQ scores were compared according to the median of sera activation, using independent samples t-test.

Results
Serum ADRA1 activation associated with the OHQ composite score (beta 0.77 OHQ points per SD of activity; p=0.009), whereas there were no significant associations among controls (p=0.953). The association between ADRA1 and total OHQ was significant also after adjusting for heart rate and systolic blood pressure at 3 min (p=0.024). The OHQ composite score was higher in with above median serum ADRA1 activation (Figure 1).
ADRA1 activation associated with a higher score for vision problems (p<0.001) and symptoms during prolonged standing (p=0.037) and walking for short (p=0.042) or long periods (p=0.001).
The activity of ARB2, CHRM2 and OPRL1 did not associate with OHQ composite score in neither POTS patients nor controls.
Figure 1. OHQ according to ADRA1 activation

Conclusions
Activating serum proteins for the adrenergic alpha-1 receptor are associated with the severity of orthostatic symptoms in POTS patients, partly independent of the orthostatic hemodynamic response. Serum activity against alpha1 receptors is related particularly to symptoms of disturbed vision and symptoms during walking or prolonged standing. Serum activity against the adrenergic alpha 1 receptor may be a one factor underlying the orthostatic symptoms in POTS.

Acknowledgement/Funding
The Swedish Heart-Lung Foundation, The Swedish Heart and Lung Association, Solidex, ALF funds, Crafoord Foundation, Ernhold Lundströms Foundation.