Human Left Ventricular 400µm Slices for Optical Imaging cut with 7000smz-2

Abstract

(from Acetylcholine Reduces IKr and Prolongs Action Potentials in Human Ventricular Cardiomyocytes, Koncz et al, 2022)

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Vagal nerve stimulation (VNS) has a meaningful basis as a potentially effective treatment for heart failure with reduced ejection fraction. There is an ongoing VNS randomized study, and four studies are completed. However, relatively little is known about the effect of acetylcholine (ACh) on repolarization in human ventricular cardiomyocytes, as well as the effect of ACh on the rapid component of the delayed rectifier K+ current (IKr). Here, we investigated the effect of ACh on the action potential parameters in human ventricular preparations and on IKr in human induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs). Using standard microelectrode technique, we demonstrated that ACh (5 µM) significantly increased the action potential duration in human left ventricular myocardial slices. ACh (5 µM) also prolonged repolarization in a human Purkinje fiber and a papillary muscle. Optical mapping revealed that ACh increased the action potential duration in human left ventricular myocardial slices and that the effect was dose-dependent. Perforated patch clamp experiments demonstrated action potential prolongation and a significant decrease in IKr by ACh (5 µM) in hiPSC-CMs. Computer simulations of the electrical activity of a human ventricular cardiomyocyte showed an increase in action potential duration upon implementation of the experimentally observed ACh-induced changes in the fully activated conductance and steady-state activation of IKr. Our findings support the hypothesis that ACh can influence the repolarization in human ventricular cardiomyocytes by at least changes in IKr.

Method

Human Myocardial Slices and Optical Imaging

Human donor hearts unsuitable for transplantation were obtained from the Washington Regional Transplant Community (WRTC). The preparation of left ventricular (LV) slices has been described previously [28]. Briefly, LV tissue was isolated, and approximately 1 cm3 cubes were cut in 4 °C cardioplegic solution and attached to the tissue holder of a vibrating microtome (7000 smz-2, Campden Instruments Ltd., Loughborough, UK). LV slices (400 μm thickness) were sectioned at 80 Hz frequency and 0.04 mm/s speed in 4 °C slicing solution containing (in mM): NaCl 140, KCl 6, MgCl2 1, CaCl2 1.8, glucose 10, HEPES 10; pH 7.4. 2,3-Butanedione (10 mM) was added to suppress contraction. Slices were then incubated for at least 20 min at room temperature in recovering solution (containing (in mM): NaCl 140, KCl 4.5, MgCl2 1, CaCl2 1.8, glucose 10, HEPES 10, BDM 10; pH 7.4) and then used in acute studies. To optically map LV slices, slices were pinned down (Minutien Pins, 26002-10, Fine Science Tools, Foster City, CA, USA) to a bath containing Tyrode’s solution at 37 °C and paced using a homemade bipolar platinum electrode (Coated Platinum-Iridium Wire, 778000, A-M Systems, Sequim, WA, USA) at 1.5× voltage threshold of stimulation, 2 ms pulse width and frequencies ranging from 0.5 Hz to loss of 1:1 capture. Blebbistatin (10–15 mM) was added to Tyrode’s solution during optical mapping to arrest motion. Slices were incubated in RH237 (voltage sensitive dye, Biotium, Fremont, CA, USA, 61018) and Rhod2-AM (calcium indicator dye, Thermo Fisher Scientific, Waltham, MA, USA, R1244) sequentially and excited with a green LED light source (520 ± 17 nm). Emitted light was collected using a tandem lens optical mapping system and recorded at 1000 Hz using two CMOS cameras (Ultima-L, SciMedia, Costa Mesa, CA, USA). Data analysis was performed using a custom MATLAB program.