Dexmedetomidine exhibits antiarrhythmic effects on human-induced pluripotent stem cell-derived cardiomyocytes through a Na/Ca channel-mediated mechanism

Introduction

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are among the most promising cell therapy candidates for several heart disease types, including myocardial infarction, which can result in heart failure. Numerous studies have shown that hiPSC-CMs can engraft and couple to the host myocardium to contribute to functional improvement (1-3). The expression of physiologically functional ion channels may play an essential role in this functional recovery process (4,5). Since hiPSC-CMs represent a potentially unlimited source of human cardiomyocytes with limited ethical concerns, they may offer an excellent opportunity to explore the physiological and pathophysiological mechanisms that drive human heart function. Furthermore, ventricular-like hiPSC-CMs exhibit the normal electrophysiological characteristics of spontaneous contractions. Therefore, they are a suitable model for studying the physiological effects and mechanisms of certain substances on cardiac tissues at the cellular level (6,7).

Dexmedetomidine (DMED) is a highly selective and potent α₂-adrenoceptor agonist with sedative, analgesic, and sympatholytic properties (8). The use of DMED is becoming increasingly popular in both pediatric and adult surgeries due to the absence of concurrent respiratory depression and opioid-sparing effects. Although DMED is not well tolerated hemodynamically and increased administrations have been shown to cause hypotension and bradycardia (9-11), these effects are generally clinically benign and self-limiting. DMED has even been shown to have a preventive effect on some types of atrioventricular tachycardia and junctional tachyarrhythmias (12). These antiarrhythmic effects of DMED are believed to be associated with the classical negative feedback mechanism modulating the catecholamine release through the activation of presynaptic α_2A-adrenoceptors expressed throughout the central nervous system (13-15). However, a study led by Pan et al. (16) revealed that DMED directly inhibited the electrophysiological effects of pacemaker cells in the sinoatrial nodes of rabbits, and this effect was independent of the α₂-adrenoreceptor. Furthermore, Knaus et al. (17) reported that clonidine, as an α₂-adrenoreceptor agonist, displayed direct inhibition of cardiac hyperpolarization-activated cyclic nucleotide-gated (HCN) pacemaker channels contributing to its bradycardic effects in the isolated spontaneously beating right atria of mice. However, the mechanisms by which DMED can directly impact spontaneously beating ventricular cardiomyocytes in humans remain elusive.

The present study aimed to investigate the electrophysiological effects and potential mechanisms of DMED on hiPSC-CMs. Herein, we report the effects of DMED on spontaneous action potentials, potassium (K⁺) channel currents (I_K1 and I_Kr), hyperpolarization-activated pacemaker currents (I_f), sodium (Na⁺) channel currents (I_Na), and calcium (Ca²⁺) channel currents (I_Ca) in hiPSC-CMs over 30 days. The goal was to explore the possible short effectively- and long-term mechanisms of cardiac inhibition by DMED. This article is presented following the Materials Design Analysis Reporting (MDAR) checklist (available at http://dx.doi.org/10.21037/atm-20-5898).

Methods

The generation and identification of hiPSC-CMs

The hiPSC-CMs were generated from human-induced pluripotent stem cells (hiPSCs), which were gifted by Professor Li Yanxin of the Shanghai Children’s Medical Center. The ethics committee approved all experiments of the Shanghai Children’s Medical Center, Shanghai Jiao Tong University (SCMCIRB-W2020007). The hiPSCs were generated as previously described (18). Briefly, episomal vectors of octamer-binding transcription factor 4 (OCT4), SRY (sex-determining region Y)-box 2 (SOX2), Kruppel-like factor 4 (KLF4), and MYC were electrotransferred into human cord blood cells to generate the hiPSCs. Cells were maintained in a Matrigel-coated system and cultured with E8 medium (05990, STEMCELL Technologies, Canada). Two days before differentiation, the hiPSCs were dissociated into single cells with Accutase (7920, STEMCELL Technologies, Canada) at 37 °C for 4 minutes and seeded into an evenly distributed monolayer onto a Matrigel-coated cell culture dish at 100,000–200,000 cell/cm². On day 0, hiPSCs were fully confluent and treated with the glycogen synthase kinase 3 (GSK3) selective inhibitor CHIR99021 (72054, STEMCELL Technologies, Canada) in Roswell Park Memorial Institute (RPMI) 1640/B27 medium without insulin (RPMI1640/B27-ins, GIBCO, 11875093/A1895601, USA), and the medium was changed on day 1. On day 3, cells were treated with the Wnt inhibitor IWP2 (72124, STEMCELL Technologies, Canada) to direct the hiPSCs into a cardiac lineage further. Starting from day 7 and after that, the cells were cultured in RPMI1640/B27 with insulin (RPMI1640/B27+ins, GIBCO, 11875093/17504044, USA), and the medium was changed every other day for the next 3 weeks. The cells were screened routinely for the presence and/or absence of lineage markers.

The differentiated hiPSC-CMs were characterized by the presence of the cardiomyocyte structural marker sarcomeric-α-actinin, and myosin light chain 2 (MLC2V) was used as a marker for ventricular cardiomyocytes. Immunofluorescence staining was performed on day 30. Briefly, the cells were fixed with 4% paraformaldehyde for 15 minutes, and then the cell membrane was permeabilized with 0.5% Triton X-100 for 20 minutes at room temperature. After fixation and permeabilization, the cells were blocked with 5% bovine serum albumin (BSA) in phosphate-buffered saline (PBS) at 37 °C for 30 minutes. The cells were then incubated with a rabbit anti-MLC-2V primary antibody (Proteintech, 10906-1-AP, USA, 1:200) and a mouse anti-α-actinin (sarcomeric) primary antibody (Sigma-Aldrich, A7811, USA, 1:200) overnight at 4 °C. This was followed by incubation with an Alexa Fluor-555 donkey anti-mouse IgG (AB150106, Abcam, 1:1,000) and an Alexa Fluor-488 donkey anti-rabbit IgG (AB150073, Abcam, 1:1,000) for 20–30 minutes in PBS at room temperature. After incubation, the cells were stained with 4’6-Diamidino-2-Phenylindole (DAPI; Beyotime Biotechnology, C1002, China, 1:1,000) for 10 minutes in PBS. Immunofluorescence imaging was performed using the Leica TSC SP8 laser confocal microscopic system (Leica, DMI6000, Germany).

To further analyze the purity of the hiPSC-CMs, on day 30, cultured cells were dissociated using the human cardiomyocyte digestion solution I/II (CELLAPY, CA2011007/CA2012008, China) according to the manufacturer’s instructions. Briefly, the digestion solution I was applied for 10 minutes, followed by applying the digestion solution II for 20 minutes. The Foxp3/Transcription Factor Staining Buffer Set (Invitrogen, 00-5523-00, USA) was then used to fix and permeabilize the cells following the manufacturer’s instructions. Briefly, cells were incubated with 200 µL fixation/permeabilization solution for 30 minutes on ice. The permeabilized cells were then incubated with a FITC-conjugated anti-cardiac troponin T antibody (1C11-FITC; Abcam, ab105439, UK, 1:100). The stained cardiomyocytes were analyzed using a flow cytometer (FACS Canto, BD, USA). Histogram plots were generated using the FlowJo software (V10.0.7, BD, USA) to quantify the percentage of live cells that were cardiac troponin T (CTNT) positive.

Electrophysiological recordings

The cultured hiPSC-CMs (4×10⁴ to 8×10⁴ cells/35 mm dish) were first digested with 1 mL 1/4X Accutase (7920, STEMCELL Technologies, Canada, diluted in PBS) for one minute. The cells were then washed once with baseline extracellular fluid and transferred to the stage of an inverted microscope (ECLIPSE Ti-U, Nikon, Japan) for patch-clamp recording. The hiPSC-CMs were continuously perfused with an extracellular solution through a “Y-tube” system with a solution exchange time of 1 minute. Drugs were applied through the “Y-tube” system for 3 minutes each time. Whole-cell patch-clamp recordings on the hiPSC-CMs were performed using Axopatch 700B (Axon Instruments, Inc., Union City, CA, USA) amplifiers under an inverted microscope at room temperature (22–25 °C). Patch pipettes were fabricated from 1.5 mm outside diameter thin-walled borosilicate glass tubing (Sutter Instruments Co, Novato, CA, USA) using the Flaming/Brown micropipette puller P97 (Sutter Instruments Co, Novato, CA, USA). The patch pipette tips’ final resistance was 2–4 MΩ after heat polish and internal solution filling. After “gigaseal” between the patch pipette and cell membranes was formed, gentle suction was used to rupture the cell membrane and to establish a whole-cell configuration. All current signals were digitized with a sampling rate of 10 kHz and filtered at a cutoff frequency of 2 kHz (Digidata 1550A, Axon Instruments, Inc., Union City, CA, USA). The spontaneous action potentials were recorded by the gap-free mode with a sampling rate of 1 kHz and filtered at a cutoff frequency of 0.5 kHz. Series resistance was compensated by 40–80%. If the series resistance was more than 10 MΩ or changed significantly during the experiments, the recordings were discarded from further analysis. For the I_Na recordings, the linear leakage currents were subtracted by using an on-line “p-5” procedure (in which the currents evoked by 5 hyperpolarizing pulses with one-fifth amplitude of the test pulse were summed and added to the current trace of interest). For the I_Ca recordings, 1.0 µM tetrodotoxin (TTX) was added to the extracellular solution to block the I_Na.

Solutions and drugs

The pipette internal solution for action potential recording contained the following in mM concentrations: KCl 150, NaCl 5, CaCl₂ 2, EGTA 5, HEPES 10, and MgATP 5 (pH 7.2, KOH). For I_Na and I_Ca recordings, the pipette internal solution contained the following in mM concentrations: CsCl 130, NaCl 5, EGTA 10, HEPES 10, and Mg-ATP 5 (pH 7.2, CsOH). For I_f recordings, the pipette internal solution contained the following in mM concentrations: NaCl 6, K-aspartate 130, MgCl₂ 2, CaCl₂ 2, EGTA 5, Na₂ATP 2, NaGTP 0.1, cAMP 0.2, and HEPES 5 (pH 7.2, KOH). For I_k1 recordings, the pipette internal solution contained the following in mM concentrations: KCl 140, MgCl₂ 1, glucose 10, MgATP 2, EGTA 5, and HEPES 5 (pH 7.2, KOH). For I_kr recordings, the pipette internal solution contained the following in mM concentrations: CsCl 140, MgCl₂ 2, glucose 10, and HEPES 10 (pH 7.4, CsOH).

Both the baseline extracellular solution and the extracellular solution for action potential recording contained the following in mM concentrations: NaCl 140, KCl 5, CaCl₂ 1, MgCl₂ 1, glucose 10, and HEPES 10 (pH 7.4, NaOH). For I_Na recordings, the extracellular solution contained the following in mM concentrations: choline chloride 100, NaCl 50, MgCl₂ 2, 4-AP 5, HEPES 10, glucose 10, CsCl 20, and CdCl₂ 0.3 (pH 7.35, HCl). For I_Ca recordings, the extracellular solution contained the following in mM concentrations: NaCl 135, KCl 5.4, CaCl₂ 1.8, MgCl₂ 1.0, NaH₂PO₄ 0.33, HEPES 10, and glucose 10 (pH 7.35, NaOH). For I_f recordings, the extracellular solution contained the following in mM concentrations: NaCl 137.7, KCl 5.4, MgCl₂ 1, CaCl₂ 1.8, HEPES 5, NaOH 2.3, glucose 10, 4-aminopyridine 2, BaCl₂ 5, and CdCl₂ 0.2 (pH 7.4, NaOH). For I_K1 recordings, the extracellular solution contained the following in mM concentrations: NaCl 136, KCl 5.4, MgCl₂ 1, NaH₂PO4 0.33, glucose 10, HEPES 10, TTX 0.01, and 4-AP 4 (pH, 7.35, NaOH). For I_Kr recordings, the extracellular solution contained the following in mM concentrations: CsCl 140, MgCl₂ 2, EGTA 10, and HEPES 10 (pH 7.2, CsOH).

DMED was purchased from Waterstone Technology (Carmel, IN, USA). Yohimbine, idazoxan, and phentolamine were purchased from Sigma-Aldrich (St Louis, USA). In this study, 1.0 µM yohimbine, 10 µM idazoxan (19), and 1.0 µM phentolamine (20) were used. All other chemical reagents were of analytical grade and purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).

Data analysis

Data were analyzed using Clampfit 10.5 and Origin 8.0 (OriginLab, Northampton, MA, USA). For the construction of the concentration-response curve, the data were fitted with the equation y = A1 + (A2-A1)/[1 + 10^^{((LOGx0-x)*p))}], in which X is the drug concentration, X0 is the drug concentration required to produce 50% inhibition of the spontaneous action potential frequency, A1 denotes the initial value, A2 denotes the final value, and p denotes the power. For the construction of the Na⁺ channel activation curves, the normalized conductance was plotted against the test pulse potentials. It was fitted to a Boltzmann function according to the following equation: G_Na/_maxG_Na = 1/[1 + exp (V_g0.5–V_g)/k_g], where _maxG_Na is the maximal value for G_Na, V_g0.5 is the potential at which G_Na is half of _maxG_Na, and k_g is the slope factor. The Na⁺ conductance (G_Na) of the membrane was calculated by using the following equation: G_Na = I_Na/(V_g - V_r), where I_Na is the peak amplitude of the I_Na, V_g is the membrane potential achieved using a step pulse, and V_r is the reversal potential for Na⁺ channels. For the construction of Na⁺ channel inactivation curves, the normalized I_Na was plotted against the conditioning pulse potentials. It was fitted to a Boltmann function according to the following equation: I/I_max = 1/[1 + exp(V_1/2–V)/k], in which V_1/2 is the membrane potential at which half-maximal channel is inactivated and k is the slope factor. The construction of the Ca²⁺ channel activation and inactivation curves were similar to that for the Na⁺ channel. The time constant of activation and inactivation was fitted to a single-exponential function [I = A₀ + A₁exp(-t/τ)], where A₀ denotes the initial value, A₁ denotes the peak value, and τ denotes the time constant.

Statistical analysis

Data values are expressed as mean ± SEM with sample sizes (n, the number of recorded cardiomyocytes). Each experiment was repeated 5–6 times in different cardiomyocytes. Each cardiomyocyte was used only one time. Statistical analysis was performed using Origin 8.0 software. One-way analysis of variance (ANOVA) was used for multiple comparisons, and paired and/or unpaired two-tailed Student’s t-test was used for evaluating the statistical significance of differences between two group means. A P value <0.05 was considered statistically significant.

Results

The generation and identification of hiPSC-CMs

The hiPSCs were differentiated into beating cardiomyocytes according to the schematic process shown in Figure 1A. Flow cytometry analysis revealed that 80.3%±0.42% (n=3) of cells differentiated from the hiPSCs were CTNT positive on day 30 of differentiation (Figure 1B). This was further confirmed by immunofluorescence staining, which demonstrated the cardiomyocyte structural marker sarcomeric-α-actinin and the ventricular cardiomyocyte marker MLC2V the differentiated cells on day 30. The majority of the hiPSC-CMs were ventricular cardiomyocytes (Figure 1C).

Figure 1 The generation and identification of hiPSC-derived cardiomyocytes. (A) A schematic diagram of the differentiation process from hiPSC into cardiomyocyte. (B) The percentage of CTNT positive cardiomyocytes measured by flow cytometry on day 30, n=3 independent biological replicates. (C) Immunofluorescence staining for α-actinin (a cardiomyocyte structural markers), and MLC2V (a marker for ventricular cardiomyocytes). Scale bar, 100 µm. hiPSC, human-induced pluripotent stem cell; CTNT, cardiac troponin T; MLC2V, myosin light chain 2.

The patch-clamp recordings of the spontaneously beating cardiomyocytes demonstrated that the action potentials were all ventricular-like shapes (Figure 2A). This is in agreement with the classification criteria of hiPSC-CMs reported by Ma and colleagues (21).

Figure 2 The dose-dependent inhibition of action potential frequency of ventricular-like hiPSC-CMs by DMED. (A) A representative spontaneous action potential trace. (B) A 1-minute recording of a typical spontaneous action potential trace after treatment with different concentrations of DMED. (C) The inhibition of spontaneous action potential frequency by DMED at various concentrations. Abscissa: concentrations of DMED. Ordinate: % inhibition of the peak frequency of the spontaneous action potential in 1 minute. Each point represents the mean ± SEM (n=6). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine; SEM, standard error of the mean.

The effects of DMED on the frequency of spontaneous action potentials of ventricular-like hiPSC-CMs

The typical spontaneous action potential traces were inhibited by the α₂-adrenoceptor agonist DMED. At concentrations of 1.0, 10, 20, 50, and 100 µM, DMED inhibited the frequency of action potentials shown by hiPSC-CMs by 6.3%±1.8%, 16.1%±3.7%, 29.1%±12.1%, 80.2%±8.4%, and 97.5%±2.5%, respectively (Figure 2B). Further analysis demonstrated that DMED dose-dependently inhibited the frequency of spontaneous action potentials of hiPSC-CMs, with an IC₅₀ value of 27.9 µM (Figure 2C). Therefore, 30 µM DMED was used for the duration of our studies.

The effects of DMED on the action potential parameters of ventricular-like hiPSC-CMs

To explore the mechanisms by which DMED decreased the frequency of spontaneous action potentials in hiPSC-CMs, we examined the resting potential, threshold potential, amplitude, and maximal diastolic potential (Figure 3A). DMED did not significantly affect the resting potential (–49.8±2.0 vs. –46.1±2.2 mV), threshold potential (–41.7±1.5 vs. 38.7±1.8 mV), amplitude (102.0±4.7 vs. 96.5±4.8 mV), or maximal diastolic potential (–67.4±2.4 vs. 63.3±2.4 mV) of the action potentials recorded from hiPSC-CMs (Figure 3B,C,D,E). However, DMED significantly prolonged the action potential duration at 90% repolarization (APD₉₀; 461.0±20.9 vs. 561.0±16.8 ms; Figure 3F).

Figure 3 The effects of DMED on the action potential parameters of ventricular-like hiPSC-CMs (resting potential, threshold potential, amplitude, maximal diastolic potential, and APD₉₀). (A) A representative recording of the action potential with or without DMED. (B,C,D,E,F) The action potential parameters with or without DMED. *, P<0.05 compared to vehicle control (n=5). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine; APD₉₀, action potential duration at 90% repolarization.

The effects of DMED on the hyperpolarization-activated pacemaker currents of hiPSC-CMs

Pacemaker currents (I_f) play a very important role in determining and maintaining the spontaneous rhythm of action potentials in cardiac sinoatrial node cells (22,23); therefore, the effects of DMED on I_f were examined. DMED did not affect the shape or amplitudes of the current/voltage (I/V) relationship curve of I_f for both the steady-state and the peak tail currents (Figure 4).

Figure 4 The effects of DMED on hyperpolarization-activated pacemaker currents of hiPSC-CMs. (A) A representative recording of pacemaker current traces with or without DMED. (B) The I/V curves of pacemaker steady state currents with or without DMED. (C) The I/V curves of pacemaker peak tail currents with or without DMED. Each point represents the mean ± SEM (n=6). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine; I/V, current/voltage; SEM, standard error of the mean.

The effects of DMED on the K⁺ channel currents of hiPSC-CMs

Potassium ion channel currents (I_k) play a crucial role in determining and maintaining the plateau stage of action potentials in cardiac ventricular cells. As such, the effects of DMED on I_k were investigated. DMED did not affect the shape or the amplitudes of the I/V/l relationship curves of I_k1 and I_kr (Figure 5).

Figure 5 The effects of DMED on the potassium channel currents of hiPSC-CMs. (A) A representative recording of the I_K1 traces with or without DMED. (B) The I/V curves of I_K1 with or without DMED. (C) The I/V curves of I_Kr with or without DMED. Each point represents the mean ± SEM (n=5). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine; I/V, current/voltage; SEM, standard error of the mean.

The effects of DMED on the Na⁺ channel currents of hiPSC-CMs

Sodium ion channels determine cardiac excitability and electrical conduction velocity. Blocking the sodium channel currents (I_Na) or slowing the action potential upstroke would suppress supraventricular and ventricular arrhythmias in the clinical setting (24). DMED inhibited the amplitude of the I_Na, but did not affect the shape of the I/V relationship curves of the Na⁺ channel (Figure 6A). The representative recordings of the I_Na in the absence or presence of DMED are shown in Figure 6B. DMED neither affected the Na⁺ channel steady-state activation curves nor the steady-state inactivation curves (Figure 6C,D). However, it significantly decreased the time constant of activation at potentials from –40 to –20 mv (Figure 6E), and significantly increased the time constant of inactivation (Figure 6F).

Figure 6 Inhibition of the hiPSC-CMs Na⁺ channels by DMED. (A) The I/V relationship curves of the Na⁺ channels in the absence or presence of DMED. (B) A representative recording in the absence or presence of DMED. (C) The Na⁺ channel activation curves in the absence or presence of DMED. (D) The effects of DMED on the steady-state inactivation of Na⁺ channels. (E,F) Data for the development of activation and inactivation were both fitted to a single-exponential function and plotted as a function of the development pulse voltage. The average time to reach the peaks and inactivation are shown. Each point represents the mean ± SEM. *, P<0.05 compared to the vehicle control (n=6). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine; SEM, standard error of the mean.

The effects of DMED on the L-Ca²⁺ channel currents of hiPSC-CMs

In our study, DMED inhibited the L type calcium ion channel current (ICa) amplitude, but it did not affect the shape of the I/V relationship curves of the Ca²⁺ channel (Figure 7A). Figure 7B illustrates the representative recordings of the I_Ca in the absence or presence of DMED. DMED did not affect the steady-state activation curves of the Ca²⁺ channel (Figure 7C), but it significantly decreased the time constant of activation at potential –20 mv (Figure 7D). Also, DMED did not affect the steady-state inactivation curves of the Ca²⁺ channel either (Figure 7E), but instead significantly increased the time constant of inactivation (Figure 7F).

Figure 7 The inhibition of hiPSC-CMs Ca²⁺ channels by DMED. (A) The I/V relationship curves of the Ca²⁺ channels with or without DMED. (B) A typical recording with or without DMED. (C) The Ca²⁺ channel activation curves with or without DMED. (D) The time constant of activation for hiPSC-CM Ca²⁺ channels. (E) The effects of DMED on the steady-state inactivation of the Ca²⁺ channels. (F) The time constant of inactivation for hiPSC-CM Ca²⁺ channels. Each point represents the mean ± SEM. *, P<0.05 compared to the vehicle control (n=5). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine; I/V, current/voltage; SEM, standard error of the mean.

The DMED-mediated inhibition of the frequency of spontaneous action potential, I_Na, and I_Ca in hiPSC-CMs was independent of the α₂-adrenoceptor, imidazoline receptor, and the α₁-adrenoceptor

DMED is a well-known, highly selective, and potent α₂-adrenoceptor agonist (14). We examined whether the α₂-adrenoceptor might be responsible for mediating the inhibitory effects of DMED on hiPSC-CMs. A dose of 10 µM yohimbine (an α2-adrenoceptor antagonist) affected the shape of the action potentials, whereas a dose of 1 µM did not, and therefore, 1 µM yohimbine was used in this study. Yohimbine could not antagonize the inhibitory effects of DMED on the frequency of spontaneous action potentials or the peak values of the I_Na and I_Ca in hiPSC-CMs. DMED in the presence of yohimbine reduced the frequency of spontaneous action potentials by 55.2%±6.2%, which was comparable to the inhibitory effects produced by DMED alone with a reduction of 52.3%±5.1% (Figure 8A). DMED in the presence of yohimbine inhibited the peak values of the I_Na and I_Ca by 45.1%±5.2% and 65.1%±4.2%, respectively, which was again similar to the inhibitory effects produced by DMED alone with reductions of 45.3%±7.1% and 63.6%±6.1%, respectively (Figure 8B,C).

Figure 8 The inhibitory effects of DMED on hiPSC-CMs were mediated through α₂-adrenoceptor-independent pathways. The I_Na was evoked by 300 ms depolarizing pulses of –70 mV to 0 mV in 10 mV increments from a holding potential of –80 mV. The I_Ca was elicited by a series of depolarizing pulses from a holding potential of –40 mV to +20 mV in 10 mV steps with a 300 ms pulse duration. Yohimbine did not affect the DMED-mediated effects on spontaneous action potential frequency (A), the I_Na (B), or the I_Ca (C) (P>0.05, n=5). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine.

DMED is not only an α₂-adrenoceptor agonist, but it is also an imidazole derivative that acts as an imidazole receptor agonist. We investigated whether the imidazole receptor could be responsible for mediating the inhibitory effects of DMED on the frequency of spontaneous action potential and the peak values of the I_Na and I_Ca in hiPSC-CMs. Idazoxan, which is both an α₂-adrenoceptor antagonist and an imidazole receptor antagonist, did not antagonize the inhibitory effects of DMED on the frequency of spontaneous action potentials (59.2%±5.2% vs. 52.3%±5.1%), nor did it affect the peak values of the I_Na (46.1%±6.2% vs. 45.3%±7.1%) and I_Ca (64.1%±5.4% vs. 63.6%±6.1%) in hiPSC-CMs (Figure 9).

Figure 9 The inhibitory effects of DMED on hiPSC-CMs were mediated through imidazoline receptor-independent pathways. Idazoxan did not affect the DMED-mediated inhibition of spontaneous action potential frequency (A), the I_Na (B), or the I_Ca (C) (P>0.05, n=5). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine.

In addition to the activation of the α₂-adrenoceptor and the imidazole receptor, DMED can also activate the α₁-adrenoceptor, although the binding capability is relatively low compared with that of the α₂-adrenoceptor. We further examined whether the α₁-adrenoceptor mediated the inhibitory effects of 30 µM DMED on the frequency of spontaneous action potential and the peak values of the I_Na and I_Ca in hiPSC-CMs. The α₁-adrenoceptor antagonist phentolamine did not antagonize the inhibitory effects of DMED on the frequency of action potentials (57.3%±7.2% vs. 52.3%±5.1%), nor the peak values of the I_Na (47.6%±5.8% vs. 45.3%±7.1%) and I_Ca (66.7%±6.4% vs. 63.6%±6.1%) in hiPSC-CMs (Figure 10).

Figure 10 The inhibitory effects of DMED on hiPSC-CMs were mediated through α₁-adrenoceptor-independent pathways. Phentolamine did not affect the inhibition of spontaneous action potential frequency (A), the I_Na (B) or the I_Ca (C) mediated by DMED (P>0.05, n=5). hiPSC-CMs, human-induced pluripotent stem cell-derived cardiomyocytes; DMED, dexmedetomidine.

Discussion

Following previously described protocols (25), about 80% of hiPSCs in this study were successfully differentiated into cardiomyocytes over 30 days. The differentiated cardiomyocytes were characterized by the presence of sarcomeric-α-actinin and MLC2V, suggesting a ventricular phenotype. The action potential amplitude, maximal diastolic potential, APD₉₀ (Figure 3), and frequency of contractions (Figure 2) recorded from the hiPSC-CMs cultured over 30 days were similar to that reported in previous studies (21,26,27). These results indicated that the hiPSC-CMs generated by our induction and differentiation protocol were comparable to previous reports and the electrophysiological characteristics of the hiPSC-CMs used in the current study also agree with previous investigations.

The hiPSC-CMs generated in this study demonstrated ventricular-like spontaneous action potentials, and both the morphological and physiological characteristics were also suggestive of ventricular cardiomyocytes. However, the previous study has shown differences in the morphological and physiological characteristics between hiPSC-CMs and human ventricular myocytes (28). It has been widely reported that hiPSC-CMs structurally resemble human fetal ventricular cardiomyocytes. Human ventricular myocytes are large and cylindrical (approximately 150 µm × 10 µm), while hiPSC-CMs are smaller (30 µm × 10 µm) (29). Also, human ventricular myocytes are bi- or multi-nucleated, whereas most hiPSC-CM are mono-nuclear. The extensive t-tubule network present in human ventricular myocytes is absent in hiPSC-CMs (30). Compared to mature ventricular myocytes, hiPSC-CMs exhibit reduced electrical excitability, impaired excitation-contraction coupling, and incomplete adrenergic sensitivity (28). However, it was reported that most ion channels present in healthy or diseased cardiomyocytes were also present in hiPSC-CMs, and in fact, some of them contribute to action potential performance and are regulated by adrenergic stimulation (31).

DMED inhibited the frequency of ventricular-like spontaneous action potentials and significantly prolonged the APD₉₀ in a dose-dependent manner. DMED had a negligible effect on the I_f, I_K1, and I_Kr. However, DMED inhibited the I_Na and I_Ca of hiPSC-CMs, and this effect was independent of the α₂-adrenoceptor, the imidazoline receptor, and the α₁-adrenoceptor. The shapes of the activation and inactivation curves of the Na⁺ and Ca²⁺ channels were not affected by DMED, but an effect was noted on time constant of activation and inactivation on these two channels.

The potential application of DMED in treating or preventing tachyarrhythmias has been reported, especially in the pediatric population (12). The antiarrhythmic effects of DMED are traditionally thought to be caused by the negative feedback modulation of catecholamine release through the activation of presynaptic α₂-adrenoceptors expressed throughout the central nervous system and enhanced vagal activity (32,33). However, recent in vitro experiments (34) showed that DMED attenuates L-calcium channel currents in dissociated ventricular myocytes from adult rat hearts and inhibits the firing rate of pacemaker cells from the sinoatrial nodes, indicating that DMED may have direct inhibitory effects on the myocardium. Our study further supports a DMED-mediated direct inhibitory effect on myocardial cells. We demonstrated that DMED dose-dependently inhibited the frequency of spontaneous ventricular-like action potentials and prolonged the APD₉₀ in hiPSC-CMs without nerve innervations (Figures 2 and 3).

The I_f contributes to the spontaneous rhythmic activities in both the heart and the brain, and this rhythmic activity is in turn encoded by the HCN family of genes consisting of the HCN1–4 genes (35). Although the I_f current stemming from the sinoatrial node is essential for the onset and control of the heart rate (36), the I_f the current from hiPSC-CMs with ventricular-like action potentials did not mediate the inhibitory effects of DMED on the frequency of spontaneous action potentials. The I_K contributes to the plateau stage of cardiac ventricular cells. Although the K_r channel is a well-known nonspecific target of a broad range of drugs, the I_Kr current from hiPSC-CMs was not involved in the DMED-mediated prolongation of APD₉₀. In this study, DMED did not affect the shape, amplitudes, and I/V relationship curves of the I_K1 and I_Kr, suggesting that DMED prolonged the APD₉₀ of hiPSC-CMs through pathways independent of I_K1 and I_Kr (Figure 5).

The cardiac I_Na contributes to the action potential upstroke in ventricular cardiomyocytes. Our study demonstrated that DMED inhibited the amplitude of I_Na, decreased the time constant of activation at the potential from –40 to –20 mv, and increased the time constant of inactivation (Figure 6). Although a great number of studies have investigated the effects of DMED on Na⁺ channels in different cells (20,37,38), it is generally thought that the blockade of Na⁺ channels is related to the high affinity of drugs to a site on the open, inactivated or resting state of Na⁺ channels (39). Indeed, our previous study showed that DMED inhibited I_Na through preferential binding to inactivated Na⁺ channels and produced a frequency-dependent blockade of Na⁺ channels in rat superior cervical ganglion (20). According to a study by Ross et al. (26), the average I_Na was about 6 nA, which was similar to our primary recording. To improve the quality of patch-clamp experiments and reduce voltage-clamp errors caused by large sodium currents, 100 mM NaCl in the extracellular solution for the I_Na recordings was replaced with 100 mM choline chloride to reduce the peak values of the I_Na.

In addition to the inhibition on the I_Na, DMED also inhibited the amplitude of the I_Ca, decreased the time constant of activation at potential –20 mv, and increased the time constant of inactivation (Figure 7). Using rat dissociated ventricular myocytes, Zhao et al. showed that DMED acted in a dose-dependent manner to inhibit the I_Ca-L and slow down the activation process of Ca²⁺ channels but not the inactivation process. Additionally, DMED extended the recovery time from inactivation of the channels to reduce Ca²⁺ inward flow. This may contribute to the negative effects on myocardial contractility and cardiac electrical activity. Furthermore, the inhibition of the I_Ca-L by DMED at a concentration of 10 ng/mL was partially reversed by yohimbine, suggesting that the inhibitory effect of DMED on the I_Ca-L is partially associated with α₂-adrenergic receptors (34). However, our study showed that DMED inhibited the I_Ca without affecting the calcium channel’s activation and inactivation curves in hiPSC-CMs (Figure 7). Furthermore, the inhibitory effect of DMED on the I_Ca was not reversed by yohimbine (Figure 8), indicating that the inhibitory effect of DMED on the I_Ca in the hiPSC-CMs was not associated with α₂-adrenergic receptors. The differences between these two studies remain to be elucidated. According to a report by Yonemizu et al., there were no T-type Ca²⁺ channels detected in the hiPSC-CMs after 38–44 days of induction and differentiation (27).

Although α₂-adrenoceptors, imidazoline receptors, and α₁-adrenoceptors were detected in adult rat ventricular cardiomyocytes and hiPSC-CMs, the DMED-mediated inhibition of the action potential frequency, Na⁺ channels, and Ca²⁺ channels were not reversed by yohimbine, idazoxan, or phentolamine, suggesting that the inhibitory effects caused by DMED in hiPSC-CMs were independent of the α₂-adrenoceptor, the imidazoline receptor, and the α₁-adrenoceptor. This contrasts with reports from Zhao et al., demonstrating that the inhibitory effects of DMED on the I_Ca-L in rat ventricular myocytes were partially mediated by α₂-adrenoceptors (34).

The explicit mechanisms by which DMED regulates ion channels have yet to be established in hiPSC-CMs. Although several methods have been adopted to enhance the maturation of hiPSC-CMs in vitro, hiPSC-CMs still demonstrate a fetal phenotype in structure and function (40,41). As hiPSC-CMs do not perfectly imitate adult human cardiomyocytes, these differences may cause inaccurate disease modeling and drug screening results that may not precisely reflect the clinical setting. It is generally accepted that hiPSC-CMs could be utilized in a “case by case” manner to carefully screen patients’ sensitivity and selectivity to certain drugs. Certain genetic compositions related to inherited genetic mutations or variations in the source cells also deserve careful consideration.

In conclusion, our results demonstrated that DMED induced inhibitory effects on the spontaneous action potential, the I_Na, and the I_Ca of hiPSC-CMs, and these effects were not related to the α₂-adrenoceptor, the imidazoline receptor, or the α₁-adrenoceptor. Although further investigations are needed to elucidate the full effects of DMED on cardiomyocytes, our novel results suggest that the inhibitory effects of DMED on hiPSC-CMs may contribute to its antiarrhythmic effects.

Acknowledgments

The authors thank Professor Li Yanxin (Shanghai Children’s Medical Center, Shanghai, China) for providing cell resources.

Funding: This study was sponsored by the National Natural Science Fund of China (81670372), the Shanghai Pujiang Program (18PJD031), the Shanghai Collaborative Innovation Center for Translational Medicine (TM201821), the National Key Research and Development Program of China (2018YFC2001904), the Biomedical Engineering Fund of Shanghai Jiao Tong University (YG2016MS28), the Shanghai Key Laboratory of Tissue Engineering, and the Pediatric Postgraduate Clinical Research Capacity Improvement Progress from the School of Medicine, Shanghai Jiao Tong University (EKKY2018003DGD).

Footnote

Reporting Checklist: The authors have completed the Materials Design Analysis Reporting (MDAR) checklist. Available at http://dx.doi.org/10.21037/atm-20-5898

Data Sharing Statement: Available at http://dx.doi.org/10.21037/atm-20-5898

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/atm-20-5898). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. All experiments were approved by the ethics committee of the Shanghai Children’s Medical Center, Shanghai Jiao Tong University (SCMCIRB-W2020007).

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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