Acetylcholine Reduces L-Type Calcium Current without Major Changes in Repolarization of Canine and Human Purkinje and Ventricular Tissue

Abstract

Vagal nerve stimulation (VNS) holds a strong basis as a potentially effective treatment modality for chronic heart failure, which explains why a multicenter VNS study in heart failure with reduced ejection fraction is ongoing. However, more detailed information is required on the effect of acetylcholine (ACh) on repolarization in Purkinje and ventricular cardiac preparations to identify the advantages, risks, and underlying cellular mechanisms of VNS. Here, we studied the effect of ACh on the action potential (AP) of canine Purkinje fibers (PFs) and several human ventricular preparations. In addition, we characterized the effects of ACh on the L-type Ca2+ current (ICaL) and AP of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and performed computer simulations to explain the observed effects. Using microelectrode recordings, we found a small but significant AP prolongation in canine PFs. In the human myocardium, ACh slightly prolonged the AP in the midmyocardium but resulted in minor AP shortening in subepicardial tissue. Perforated patch-clamp experiments on hiPSC-CMs demonstrated that 5 µM ACh caused an ≈15% decrease in ICaL density without changes in gating properties. Using dynamic clamp, we found that under blocked K+ currents, 5 µM ACh resulted in an ≈23% decrease in AP duration at 90% of repolarization in hiPSC-CMs. Computer simulations using the O’Hara–Rudy human ventricular cell model revealed that the overall effect of ACh on AP duration is a tight interplay between the ACh-induced reduction in ICaL and ACh-induced changes in K+ currents. In conclusion, ACh results in minor changes in AP repolarization and duration of canine PFs and human ventricular myocardium due to the concomitant inhibition of inward ICaL and outward K+ currents, which limits changes in net repolarizing current and thus prevents major changes in AP repolarization.

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“…The absence of AP repolarization changes is in line with the work of Veerman et al [22] and Li et al [23], who found that CCh did not lead to major alterations in APD in RA-treated hiPSC-CMs, except when I K,ACh is largely increased by a GNB5 variant [32,33]. In contrast, muscarinic receptor activation in human atrial tissue [53,54] and freshly isolated human isolated cardiomyocytes [55] results in an AP shortening. The exact reason for this discrepancy is unknown, but it may be related to the still somewhat immature electrophysiological properties of hiPSC-CMs [56] and potential differences in I K,ACh density between hiPSC-CMs (Figure 2) and human atrial cells [55].…”

Injection of IK1 through dynamic clamp can make all the difference in patch-clamp studies on hiPSC-derived cardiomyocytes

“…According to Eq. 1 , the injected I K1 exhibits a peak outward amplitude of 2 pA/pF, as routinely used in our laboratory (see, e.g., Veerman et al, 2016 ; Hilderink et al, 2020 ; Knottnerus et al, 2020 ; Verkerk et al, 2021a ; Koncz et al, 2022 ; Verkerk et al, 2022 ). Such I K1 amplitude results in quiescent hiPSC-CMs with a resting membrane potential near −80 mV.…”

“…These days, the dynamic clamp technique is routinely used in our patch-clamp laboratory. We have published several papers on the dynamic clamp technique per se ( Wilders, 2006 ; Berecki et al, 2014 ; Verkerk et al, 2017 ; Verkerk and Wilders, 2021 ) as well as on studies in which the dynamic clamp technique was used as a tool in patch-clamp experiments on cultured or freshly isolated atrial cardiomyocytes ( Majumder et al, 2020 ; Verkerk et al, 2021b ) or on hiPSC-CMs ( Meijer van Putten et al, 2015 ; Veerman et al, 2016 ; Portero et al, 2017 ; Veerman et al, 2017 ; Eroglu et al, 2020 ; Hilderink et al, 2020 ; Knottnerus et al, 2020 ; Verkerk et al, 2021a ; Eroglu et al, 2021 ; Marchal et al, 2021 ; Koncz et al, 2022 ; Portero et al, 2022 ; Verkerk et al, 2022 ; Nasilli et al, 2023 ) to supply these cells with a synthetic I K1 during AP measurements.…”

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“…The cells were then detached from the surface by pipetting, the enzyme solution was diluted with addition of RPMI/B27− medium and the cells in suspension were collected by centrifugation. The cell pellet was further dissociated in presence of Liberase TM Research Grade (Roche/Merck) as previously reported 40 . The hiPSC-CMs, seeded on Matrigel Matrix-coated cover glasses (VWR International GmbH) in RPMI 1640/B27− medium, were analysed 8-10 days after dissociation.…”