LAUSR

Abnormal activity of muscarinic receptor (M-receptor) activated potassium current (IK,ACh) has been suggested as a potential atrial-specific drug target for antiarrhythmic therapy in patients with atrial fibrillation (AF). Recent work suggests that atrial myocytes derived from human induced pluripotent stem cells (iPSC-CM) hold great potential for in vitro disease modeling and patient-specific drug testing. Here we aim to investigate functional expression of IK,ACh channels in these cells. Atrial differentiation was initiated by temporal WNT and retinoic acid signaling modulation. Action potentials (APs) were recorded using sharp electrode technique in engineered heart muscle preparations (EHM). Currents were measured with voltage-clamp technique in atrial and ventricular iPSC-CM. IK,ACh was activated with a saturating concentration of the non-selective M-receptor agonist carbachol (CCh, 2 μM). APs measured in atrial EHMs were shorter compared to their ventricular counterparts (AP duration at 90% repolarization [APD90]: 163.2±3.7 ms, n=6 [EHMs] vs. 210.2±3.1 ms, n=3, p<0.001). In addition, application of CCh resulted in APD90 reduction by ∼18% and hyperpolarisation of the resting membrane potential in atrial but not in ventricular EHMs, pointing to functional IK,ACh expression in atrial preparations. Basal inward-rectifier current, (at −100 mV) in the absence of muscarinic receptor agonists was larger in ventricular iPSC-CM compared to atrial differentiation (−26.1±3.8 pA/pF, n=82/5 [n = cells/line] vs. −15.6±1.6 pA/pF, n=71/5, p<0.02.). CCh-induced current increase, attributed to IK,ACh, was detectable in all myocytes from atrial preparations (−25.2±3.6 pA/pF, n=71/5). The time course of activation with a slow decrease in current amplitude during the continuous presence of CCh resembled measurements in freshly isolated human atrial myocytes. In ventricular iPSC-CM preparations only 13 out of 73 myocytes showed an increase in inward-rectifier current in response to CCh, suggesting that ventricular preparations may contain a minor fraction of atrial myocytes. Of note, in the presence of the selective IK,ACh blocker tertiapin (100 nM), CCh had no effect on basal inward-rectifier current, providing further evidence for the molecular identity of the CCh-induced current increase as IK,ACh. Interestingly, TTP reduced basal current amplitude in CM isolated from atrial EHMs by ∼54% suggesting that agonist-independent, constitutively active IK,ACh contributes to the basal inward-rectifier current amplitude in these myocytes. Our data demonstrate that atrial iPSC-CMs express functional IK,ACh-channels, which can be activated by M-receptor agonists. These findings further outline the potential of atrial iPSC-CMs and EHMs as a promising tool for the investigation of channel-associated pathologies such as AF. grants from the DFG (VO 1568/3-1, IRTG1816, and SFB1002 project A13), the Else-Kröner-Fresenius Foundation (EKFS 2016_A20) and the DZHK (DZHK GOE MD3)