Human Cardiomyocytes Obtained by Directed Differentiation of Human Induced Pluripotent Stem Cells as Isoprenaline-Based Model to Evaluate Arrhythmogenicity

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Introduction

Isoprenaline is a non-selective beta-adrenergic agonist indicated for the treatment of heart block, Adams-Stokes attacks, bronchospasm during anesthesia, kadiakia arrest, hypovolemic shock, septic shock, hypoperfusion, congestive heart failure, and cardiogenic shock [Kislitsina et al., 2019; Szymanski, Singh, 2024].

Isoprenaline is indicated for the treatment of mild or transient episodes of heart block not requiring electrical shock or pacemakers, serious episodes of heart block and Adams-Stokes attacks not caused by ventricular tachycardia or fibrillation, and bronchospasm during anesthesia [Kislitsina et al., 2019]. Isoprenaline is also indicated in cardiac arrest until more effective treatments such as electric shock and pacemakers become available.

Adrenergic receptor (AR) stimulation results in an increase in heart rate, conduction velocity, and changes in the strength of muscle contractions, i.e. it is a compensatory mechanism that temporarily maintains cardiac output and thus sufficient organ perfusion and tissue oxygen supply. However, in pathologies such as heart failure, ventricular adrenoceptor stimulation may lead to arrhythmias. Potentially arrhythmogenic mechanisms are intracellular mechanisms when adrenaline acts on cardiac tissue. Adrenoceptor stimulation has different effects on the duration of repolarization in epicardial and endocardial cells [Akar and Rosenbaum, 2003]. Chronic stimulation of the AR promotes molecular and structural changes including pathological hypertrophy [van Berlo et al., 2013], cardiac fibrosis [Bacmeister et al., 2019], and inflammation [Murray et al., 2000] with deteriorating consequences for cardiac function.

In modern medicine, the use of β-blockers is an integral part of the therapy of heart failure [Wachter et al., 2012]. Despite significant progress in understanding the role of β-adrenergic signaling in heart disease and arrhythmias, a quantitative and functional understanding of the role of autonomic stimulation in normal cardiac electrophysiology and life-threatening arrhythmias is still incomplete [Grandi and Ripplinger, 2019].

The two main subtypes of β-adrenergic receptors are β1- and β2-adrenergic receptors. β1-adrenergic receptors account for approximately 80% and initiate a general cellular response, while β2-adrenergic receptors are grouped in the caveolar domain, in the T-tubule region [Nikolaev et al., 2009]. β2-adrenergic receptors are associated predominantly with L-type Ca2+ channels [Bristow et al., 1986]. Recent studies have shown the effectiveness of β2-blockers and non-selective β-blockers, which demonstrates the importance of β2-receptors in the development of heart failure [Ahmed, 2022; Rahm et al., 2023]. The potentially arrhythmogenic role of β2-receptors is the formation of delayed afterdepolarization [Lang et al., 2015]. β3-adrenergic receptors are expressed to a much lesser extent, and their function in the heart is poorly understood [Cannavo, Koch, 2017]. In the human ventricle, β3-adrenergic receptors can mainly counteract the effects of β1- and β2-adrenergic receptor activation.

Stimulation of β-adrenergic receptors on cardiomyocytes triggers a signaling cascade leading to an increase in cAMP, subsequent activation of protein kinase A and phosphorylation of multiple targets both in the contractile apparatus of cells and in their conduction system, including L-type Ca channels, ryanodine receptors and myofilament proteins, including troponin I. All of these proteins provide a link between cell excitation and contraction by increasing the amount of intracellular calcium during each systole (to enhance contraction) and decreasing the sensitivity of myofilaments to calcium ions (to accelerate relaxation) [Bers, 2002]. cAMP also directly binds to hyperpolarization-activated cyclic nucleotide-gated channels, which are predominantly expressed in cardiac nodal cells, resulting in an increase in the If current, which contributes to an increase in heart rate. Isoproterenol is a pure non-selective β-adrenergic receptor agonist [Reyes et al., 1993].

Experiments on human cardiac tissue are rare, the vast majority of studies use cardiomyocytes from various animals as experimental models, while the contribution and dynamics of various ion currents can differ significantly from that in humans [Jost et al., 2013]. In this work, cells obtained by differentiation from healthy human induced pluripotent stem cell line m34sk3 were chosen as human cardiomyocytes.

iPSCs derived from somatic cells by reprogramming retain the genetic background of the donor, making them an invaluable model for investigating the genetic factors underlying cardiac electrophysiology and arrhythmogenicity. Using direct differentiation protocols, researchers can generate cardiomyocytes that closely mimic the electrophysiological properties of human ventricular myocytes.

The primary objective of this study was to quantify the effects of isoprenaline on human ventricular ion channels (INa, ICa, L, and IKs) and provide a genetically relevant dataset for modeling cardiac responses to sympathetic stimulation. By integrating human-specific data, this work contributes to bridging the gap between basic research and clinical applications, particularly in the context of genetic studies of arrhythmogenic disorders.

​Materials and Methods

The effect of isoprenaline (isoprenaline hydrochloride, Sigma Aldrich) was investigated in this work.

Human cardiomyocytes were obtained from patient-specific induced pluripotent stem cells m34sk3 by directed differentiation using an adapted GIWI protocol [ Lian et al., 2013; Burridge et al., 2014].

Ion channel currents in single isolated cardiomyocytes were recorded by patch-clamp in the “whole cell” configuration. Amphotericin B was used as a perforating agent, which was brought to a concentration of 0.24 mg/ml. Experiments were carried out at a physiological temperature of 37 °C.

The extracellular solution used for recording Na+ current consisted of 20 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 120 mM CsCl2, 10 mM D-glucose, 10 mM HEPES, 0.002 mM Nisoldipine, 0.003 ivabradine (pH 7.4 was adjusted with CsOH). The intracellular solution for recording Na+ current: 135 mM CsCl2, 10 mM NaCl, 2 mM CaCl2, 5 mM EGTA, 5 mM MgATP, 10 mM HEPES (pH 7.2 with CsOH). Chamber solution for recording Ca2+ current: 160 mM TEA-Cl, 5 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, 10 mM HEPES (pH 7.4 with CsOH). Pipette solution contained 145 mM CsCl2, 5 mM NaCl, 5 mM EGTA, 10 mM HEPES/NaOH, 5 mM MgATP (pH 7.2 with CsOH). IKs chamber solution 150 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 15 mM D-glucose, 1 mM Na-pyruvate, 0.001 mM Nisoldipine, 0.001 mM E-4031, 15 mM HEPES (pH 7.4 with NaOH). IKs intracellular solution 20 mM KCl, 5 mM MgATP, 10 mM EGTA, 125 mM K-Aspartate, 1 mM MgCl2, 2 mM Na2-Phosphocreatine, 2 mM Na2-GTP, 5 mM HEPES/KOH (pH 7.2 with KOH). The pipettes were pulled from borosilicate glass in a puller. After the formation of a gigaohm resistance (GΩ), the capacitive components were compensated using an amplifier. After compensation of the Ra resistance, voltage-gated ion channels and action potential formation were detected using the established stimulation protocols. Series resistance was compensated if necessary.

Recording of Na-channel currents was performed using a stimulating step protocol from -80 to 15 mV for 50 ms. To record the L-type Ca2+ current, a step protocol from -40 to 50 mV for 300 ms was used with a prepulse from the maintained potential from -80 to -40 mV for 100 ms. The peak ICa,L was measured at 0 mV. The slow (IKs) component of the delayed rectifier potassium current was obtained by a depolarizing pulse from -40 to +50 mV with a step of 10 mV and a duration of 2.5 s. (with a maintained potential of -70 mV). Membrane capacitance in the range from 10 to 30 pF was measured using pCLAMP10.2 software.

Data analysis was performed using Clampfit 10.2 (Molecular Devices, USA) and Origin Pro 8.1 (Originlab Corporation, USA). Averaging is performed from at least three different cardiomyocytes. Statistical processing was performed using t-test. For all results, the differences were at the level of p < 0.05.

Results

We recorded the currents of the fast sodium channel INa, the L-type calcium channel ICa, L and the slow potassium channel IKs of human cardiomyocytes differentiated from healthy iPSCs.

The fast sodium current was recorded using a stimulation protocol from -80 to +15 mV with a step of 5 mV and a duration of 50 ms. An example of currents in the control and after the action of isoprenaline (ISO) is shown in Figure 1A (the current maxima were recorded from each record in one cell). From the obtained data, after normalization to cell capacity and averaging, a voltammetric curve was constructed (Figure 1B). The amplitude of the INa current after the addition of ISO increased by 73 ± 25% compared to the control (Figure 1C). Then, the activation curves m were constructed for the control and the substance (Figure 1D). V1/2 for the control was -41.04 ± 1.15 mV, after the addition of ISO -46.05 ± 1.33 mV. Under the action of ISO, a shift in the activation curve m to the left is observed, the shift in half-height is ~ 5 mV.

Figure 1. A. Currents of the fast sodium channel INa of human cardiomyocytes differentiated from iPSCs in the control (black curve) and after the action of 1 μM ISO (blue curve); B. Volt-ampere curves I-V for INa in the control (black curve, n = 12), after the addition of ISO (blue curve, n = 9); C. Activation curve m for fast sodium channels of human cardiomyocytes differentiated from iPSCs (black curve, n = 12), after ISO (blue curve, n = 9); G. Histogram of changes in cardiomyocyte amplitude as a percentage in the control (black column, n=12) and after ISO (blue column, n=9, p ≤ 0.009)

The stimulation protocol for the current of L-type calcium channels ICa,L has the form of steps from -40 to +50 mV with a step of 10 mV and a duration of 300 ms. By normalizing the currents on the capacitance and averaging the data, volt-ampere curves were constructed (Fig. 2). The amplitude of the current ICa,L after the action of ISO increased by 120% compared to the control (p ≤ 0.004). The normalized averaged amplitudes themselves were: in the control -18.78 ± 4.32 pA/pF, after ISO -41.47 ± 3.43 pA/pF. Also after adding ISO, a shift of the volt-ampere curve to the left by ~6 mV is observed.

Figure 2. A. Recording of currents of the L-type calcium channel ICa,L of human cardiomyocytes differentiated from iPSCs in the control (black curve) and after the action of 1 μM ISO (blue curve); B. Volt-ampere curves I-V for ICa,L in the control (black curve, n = 6), after adding ISO (blue curve, n = 4); C. Histogram of the change in amplitude as a percentage in the control (black column, n = 6) and after ISO (blue column, n = 4, p ≤ 0.004)

To record the currents of slow potassium channels IKs, a stimulation protocol from -30 to +60 mV with a step of 15 mV and a duration of 2.5 s was used. An example of such currents is shown in Figure 3A. Next, a current-voltage curve was constructed for the control and after the addition of ISO (Fig. 3B). The current amplitude was normalized to the cell capacity, and at +60 mV in the protocol, the maximum current density IKs was 3.50 ± 0.49 pA / pF in the control, and 6.94 ± 0.42 pA / pF in the presence of ISO. Based on the histogram of the normalized amplitude in %, the amplitude of the IKs current increased by ~ 98% after the introduction of ISO.

Figure 3. A. Currents of the slow potassium channel IKs of human cardiomyocytes differentiated from iPSCs in the control (black curve) and after the action of 1 μM ISO (blue curve); B. Current-voltage curves I-V for IKs in the control (black curve, n=3), after adding ISO (blue curve, n=3); C. Histogram of the change in amplitude as a percentage in the control (black column, n=3) and after ISO (blue column, n=3, p≤0.008)

Discussion

This study demonstrated a strong increase in voltage-gated channel currents in human cardiomyocytes under beta-adrenergic stimulation.

One of the limitations of the study is that the maturity level of cardiomyocytes differentiated from the iPSC line may not be equivalent to real adult human cardiomyocytes [Karbassi et al., 2020]. However, in our laboratory's experience, this line reaches its electrophysiological maturity by day 30, which was shown by a stable value of the excitation wave propagation velocity and the response to periodic stimulation in the range of physiological values ​​[Slotvitsky et al., 2018].

Another limitation is that the currents were recorded at a physiological temperature of 37 °C. Measuring the INa current is difficult due to its high amplitude and fast kinetics, especially at physiological temperature. In another study, the authors showed that this can lead to a systematic error in the measurements of the activation/inactivation parameters of INa, which can be taken into account using mathematical modeling of experimental artifacts [Abrasheva et al., 2024]. Note that this study presents native current values ​​without model processing, and further interpretation using mathematical modeling is a separate task.

Modulation of L-type calcium channels after the addition of isoprenaline increased the amplitude of the ICa, L current by 120%, and the maximum of the voltammetric curve shifted to the left by 6 mV. Such an increase in the ICa, L current, together with other factors, causes an increase in calcium ions in the cytosol, which increases the contractility of the myocardium [Bers, 2002]. Reactivation of ICaL increases the susceptibility to early afterdepolarization, which is an important cause of lethal ventricular arrhythmias in long QT syndromes and heart failure [Weiss et al., 2010].

The fast sodium channel current INav under the influence of isoprenaline increased by ~73%, while the activation curve shifted to the left by 5 mV. At the moment of beta-adrenergic stimulation, protein kinase A-dependent phosphorylation enhances the INa current due to changes in conductivity [Zhou et al., 2000; Zhou et al., 2002]. This may contribute to the sympathetically mediated increase in conduction velocity and the formation of recurrent arrhythmias after myocardial infarction, which is often characterized by myocardial depolarization [Nattel et al., 2007].

The slow potassium channels IKs increased the current amplitude by 98% after the addition of isoprenaline. Under normal conditions, the density of the slow component of potassium channels IKs is lower than that of the fast component IKr in humans and other large mammals [Jost et al., 2007]. However, it is known that stimulation of β-adrenergic receptors increases IKs, while having virtually no effect on IKr [Banyasz et al., 2014], and this counteracts the increase in ICaL, preventing an increase in AP length. Indeed, physical exercise and stress are typical triggers of arrhythmia in congenital long QT syndrome type 1 (LQTS), associated with a loss of IKs function [Schwartz et al., 2001], and can be prevented by β-adrenergic receptor blockade [Vincent et al., 2009]. Computer modeling has shown that increasing the IKs/IKr ratio without changing the AP length limits the occurrence of early afterdepolarizations. It is possible that IKs is more effective in stabilizing the AP duration and suppressing early afterdepolarizations than IKr [Devenyi et al., 2017].

Note that the difference in kinetics between the more rapid activation of ICa, L and the slower increase in IKs during β-adrenergic receptor activation transiently disrupts the balance of inward and outward currents [Liu et al., 2012]. The observed imbalance can temporarily prolong the AP and favor early afterdepolarizations, as shown by computer modeling [Xie et al., 2013]. Moreover, more rapid activation of ICa, L (compared to IKs) during rapid stimulation of β-adrenergic receptors transiently increases AP restitution, which leads to the decay of reentry and accelerates the transition from ventricular tachycardia to ventricular fibrillation [Xie et al., 2014].

Conclusion

It has been established that already at an isoprenaline concentration of 1 μM there is a significant increase in the potential-dependent ion currents INav, ICa, L and IKs of human cardiomyocytes obtained by differentiation of induced human pluripotent stem cells. It has been shown that the difference in kinetics between the faster activation of ICa, L and the slower increase in IKs during activation of β-adrenergic receptors temporarily disrupts the balance of incoming and outgoing currents, which can prolong AP and favor early postdepolarization, which is a factor in the development of life-threatening arrhythmias.

 

 

About the authors

Sandaara Kovalenko

Email: sandaara.romanova@phystech.edu

Sheida Frolova

Email: sh.frolova@monikiweb.ru

serafima Romanova

Email: scherbina_serafima@mail.ru

Valeriya Tsvelaya

Email: vts@yandex.ru

Roman Syunyayev

Email: roman.syunyaev@gmail.com

Konstantin Agladze

Moscow Regional Clinical Research Institute named after M.F. Vladimirskii

Author for correspondence.
Email: agladze@yahoo.com
ORCID iD: 0000-0002-9258-436X

Doctor of Medical Sciences, Professor, Head of Researcher, Laboratory of Molecular Cell Diagnostics, Leading Researcher

Russian Federation

References

Supplementary files