Human cardiomyocytes obtained by directed differentiation of human induced pluripotent stem cells as isoprenaline-based model to evaluate arrhythmogenicity

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Background

Stimulation of adrenoceptors increases heart rate, conduction velocity, and myocardial contractility, serving as a compensatory mechanism that temporarily maintains cardiac output and ensures adequate organ perfusion and oxygen delivery to tissues. However, in pathologic conditions such as heart failure, adrenergic stimulation of the ventricles may induce arrhythmias. Intracellular mechanisms triggered by epinephrine exposure in cardiac tissue are potentially arrhythmogenic. Adrenergic stimulation has a differential effect on repolarization duration in epicardial and endocardial cells [1]. Persistent adrenoceptor stimulation promotes molecular and structural changes in cardiomyocytes and the extracellular matrix, including pathologic hypertrophy [2], cardiac fibrosis [3], and inflammation [4], which progressively impair cardiac function.

In contemporary medicine, β-blockers are an essential component of heart failure therapy [5]. Despite advances in understanding the role of β-adrenergic signaling in heart disease and arrhythmias, quantitative and functional knowledge of the role of autonomic stimulation in normal cardiac electrophysiology and in the development of life-threatening arrhythmias remains incomplete [6].

The two main subtypes of β-adrenoceptors are β₁- and β₂-adrenoceptors. β₁-adrenoceptors account for approximately 80% and initiate a whole-cell response, whereas β₂-adrenoceptors are clustered within the caveolar domain in the region of the T-tubules [7]. β₂-adrenoceptors are associated primarily with L-type Ca2+ channels [8]. Recent studies have demonstrated the efficacy of β₂-blockers and nonselective β-blockers, underscoring the importance of β₂-receptors in the development of heart failure [9, 10]. The potentially arrhythmogenic role of β₂-receptors lies in the generation of delayed afterdepolarization [11]. β₃-adrenoceptors are expressed to a much lesser extent, and their function in the heart is poorly understood [12]. In the human ventricle, β₃-adrenoceptors may primarily counteract β₁- and β₂-adrenoceptor activation effects.

Stimulation of β-adrenoceptors on cardiomyocytes initiates a signaling cascade leading to an increase in cyclic adenosine monophosphate, subsequent activation of protein kinase A, and phosphorylation of multiple targets in the contractile apparatus and conduction system, including L-type calcium channels (ICa, L): Ca, ryanodine receptors, and myofilament proteins such as troponin I. These proteins mediate excitation–contraction coupling by increasing intracellular calcium during each systole (enhancing contraction) and decreasing myofilament sensitivity to calcium ions (accelerating relaxation) [13]. Cyclic adenosine monophosphate also binds directly to hyperpolarization-activated, cyclic nucleotide–gated channels, predominantly expressed in cardiac nodal cells. As a result, the If current (inward current of the hyperpolarization-activated Na⁺/K⁺ channel) increases, promoting an elevated heart rate.

Isoprenaline[1] is a pure, nonselective β-adrenoreceptor agonist [14]. The drug is widely used in clinical practice for treating various cardiovascular disorders, including heart block, Morgagni–Adams–Stokes syndrome, and for relief of bronchospasm during anesthesia[2] [15]. In addition to its therapeutic effects, isoprenaline is of considerable interest as a tool for studying the mechanisms of β-adrenoreceptor stimulation and its role in the development of arrhythmogenic responses in cardiomyocytes.

Experiments on human cardiac tissue are rare; in most studies, animal cardiomyocytes are used as experimental models, whereas the contribution and properties of various ionic currents may differ substantially from those in humans [16]. In this work, human cardiomyocytes were derived by differentiation of induced pluripotent stem cells (iPSCs) from a healthy donor line, m34sk3. iPSCs, obtained from somatic cells through reprogramming, retain the donor’s genetic background, making them a valuable model for investigating genetic factors underlying cardiac electrophysiology and arrhythmogenicity. Using directed differentiation protocols, cardiomyocytes can be generated that closely replicate the electrophysiological properties of human ventricular myocytes.

Aim

The work aimed to investigate the effect of isoprenaline on voltage-gated ion channels in human ventricular cardiomyocytes derived from directed differentiation of iPSCs from a healthy donor, to substantiate a genetically relevant basis for modeling human cardiac electrical activity under conditions of sympathetic stimulation.

Methods

The work examined the action of isoprenaline (isoprenaline hydrochloride; Sigma-Aldrich, USA).

Human cardiomyocytes were obtained from iPSCs of the patient-specific m34sk3 line by directed differentiation using a modified GIWI protocol [17, 18]. The m34sk3 iPSC line was provided by the E.N. Meshalkin National Medical Research Center. This iPSC line was generated from cells donated by a patient without cardiovascular disease.

Ion channel currents were recorded using a patch-clamp setup consisting of the following main components: Digidata 1440A digitizer (Axon Instruments, USA), Axopatch 200B amplifier (Axon Instruments, USA), MP-285 micromanipulator (Sutter, Germany), Olympus IX71 inverted microscope (Olympus, Japan), HumBug noise eliminator (Digitimer Ltd, UK), and AVTT75 antivibration platform (Kinetic Systems, USA) [19]. The temperature of the extracellular solution in the chamber was maintained using a TC-324C automatic temperature controller (Warner Instruments, USA). Pipettes were fabricated using a P-97 micropipette puller (Sutter, Germany), borosilicate glass capillaries B150-86-10 (Sutter, Germany), and an MF-900 microforge (Narishige, Japan). The following solutions were used:

• Extracellular solution for Na+ current recording: 20 mM NaCl, 1 mM MgCl2, 1.8 mM CaCl2, 120 mM CsCl, 10 mM D-glucose, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 0.002 mM nisoldipine1, and 0.003 mM ivabradine; pH adjusted to 7.4 with cesium hydroxide;
• Intracellular solution for Na+ current recording: 135 mM CsCl, 10 mM NaCl, 2 mM CaCl2, 5 mM ethylene glycol-bis(2-aminoethyl ether)-N,N,Nʹ,Nʹ-tetraacetic acid (EGTA), 5 mM magnesium adenosine triphosphate (MgATP), and 10 mM HEPES; pH adjusted to 7.2 with cesium hydroxide;
• Bath solution for Ca2+ current recording: 160 mM tetraethylammonium chloride (TEA-Cl), 5 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES; pH adjusted to 7.4 with cesium hydroxide;
• Pipette solution: 145 mM CsCl, 5 mM NaCl, 5 mM EGTA, 10 mM HEPES/sodium hydroxide, and 5 mM MgATP; pH adjusted to 7.2 with cesium hydroxide;
• Bath solution for the slow component of the delayed rectifier potassium current (IKs): 150 mM NaCl, 5.4 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 15 mM D-glucose, 1 mM sodium pyruvate, 0.001 mM nisoldipine, 0.001 mM E-4031, and 15 mM HEPES; pH adjusted to 7.4 with sodium hydroxide;
• Intracellular solution for IKs: 20 mM KCl, 5 mM MgATP, 10 mM EGTA, 125 mM potassium aspartate, 1 mM MgCl2, 2 mM Na2 phosphocreatine, 2 mM Na2 guanosine triphosphate, and 5 mM HEPES/potassium hydroxide; pH adjusted to 7.2 with potassium hydroxide.

Action potential (AP) formation was recorded in an extracellular solution containing 150 mM NaCl, 1.8 mM CaCl2, 5.4 mM KCl, 1 mM MgCl2, 15 mM HEPES, 15 mM D-glucose, and 1 mM sodium pyruvate (pH adjusted to 7.4 with sodium hydroxide). The intracellular solution for AP recording consisted of 5 mM NaCl, 2 mM CaCl2, 150 mM KCl, 5 mM MgATP, 5 mM EGTA and 10 mM HEPES (pH adjusted to 7.2 with potassium hydroxide). For all solutions, chemically pure reagents (≥ 99.9%) from Helicon (Russia) and PanEco (Russia) were used. Solutions were filtered through 0.22-µm membrane filters (Merck Millipore, Germany).

Ion channel currents in single, isolated cardiomyocytes were recorded using the patch-clamp technique in the whole-cell configuration. Amphotericin B (Sigma-Aldrich, USA) at a final concentration of 0.24 mg/mL was used as the perforating agent [19]. Experiments were performed at the physiological temperature of 37 °C. Patch pipettes were pulled from borosilicate glass using a puller. After establishing a gigaohm seal (GΩ), capacitive components were compensated using the amplifier. Following compensation of the access resistance (Ra), voltage-gated ion channels were detected and APs were generated using preset stimulation protocols. Series resistance was compensated as required.

Na⁺ channel currents were recorded using a step stimulation protocol from −80 to 15 mV for 50 ms. For Ca2+ L-type current recording, a step protocol from −40 to 50 mV for 300 ms was applied, with a prepulse from the holding potential of −80 to −40 mV for 100 ms. ICa, L peaks were measured at 0 mV. The slow component (IKs) of the delayed rectifier K⁺ current was obtained using a depolarizing pulse from −40 to +50 mV in 10-mV increments for 2.5 s (from a holding potential of −70 mV). APs were recorded using a 1-nA current stimulus with a 2.5-ms duration. Membrane capacitance in the range of 10–30 pF was measured using pCLAMP 10.2 software (Molecular Devices, USA).

Data analysis was performed using Clampfit 10.2 (Molecular Devices, USA) and OriginPro 8.1 (OriginLab Corporation, USA). Averaging was based on measurements from at least 3 different cardiomyocytes. Statistical analysis was conducted using the Student’s t-test. Differences were considered statistically significant at p < 0.05.

Results

In human ventricular cardiomyocytes obtained by differentiation from iPSCs, the AP for these cardiomyocytes was first examined using the patch-clamp technique with a current stimulus of 1 nA and a duration of 2.5 ms [19]. The AP duration at 80% repolarization was 350 ± 14 ms (n = 7). A representative AP trace is shown in Fig. 1.

 

Fig. 1. Example of the action potential pattern of human ventricular cardiomyocytes obtained by directed differentiation of human-induced pluripotent stem cells from a healthy donor. The zero potential level is indicated by the horizontal line.

 

Next, INaV, ICa, L, and IKs currents were recorded in human cardiomyocytes differentiated from iPSCs obtained from a healthy donor [19].

The fast sodium current was recorded using a stimulation protocol from −80 to +15 mV in 5 mV increments with a duration of 50 ms. An example of current traces under control conditions and after isoprenaline application is shown in Fig. 2,a (maximal currents from each trace in a single cell are presented). From the obtained data, after normalization to cell capacitance and averaging, the current-voltage curve was constructed (Fig. 2, b). The amplitude of INaV after isoprenaline application increased by 73%± 25% compared with control (Fig. 2, d). Subsequently, activation (m) curves for the control and drug conditions were plotted (Fig. 2, c). The half-activation potential (V1/2) was −41.04 ± 1.15 mV under control conditions and −46.05 ± 1.33 mV after isoprenaline application. Under the influence of isoprenaline, a leftward shift of the m-activation curve was observed, with the half-maximum shifted by approximately 5 mV.

 

Fig. 2. Effects of isoprenaline on fast sodium channels (INaV) of human ventricular cardiomyocytes: a, INaV currents in human cardiomyocytes differentiated from induced pluripotent stem cells under control conditions (black trace) and after exposure to 1 µM isoprenaline (blue trace); b, current–voltage (I–V) relationships for INaV under control conditions (black trace, n = 12) and after isoprenaline (blue trace, n = 9); c, activation gating variable m curve for fast sodium channels in human cardiomyocytes differentiated from induced pluripotent stem cells under control conditions (black trace, n = 12) and after isoprenaline (blue trace, n = 9); d, histogram of amplitude changes in cardiomyocytes, percent under control conditions (black bar, n = 12) and after isoprenaline (blue bar, n = 9) (p ≤ 0.009). V indicates potential measured in millivolts (mV).

 

The stimulation protocol for the ICa, L current consisted of voltage steps from −40 to +50 mV in 10 mV increments with a duration of 300 ms. From the obtained data, after normalization of the currents to cell capacitance and averaging, current-voltage curves were plotted (see Fig. 3). The amplitude of ICa, L after isoprenaline application increased by 120% compared with the control (p ≤ 0.004). The normalized mean amplitudes were −18.78 ± 4.32 pA/pF under control conditions and −41.47 ± 3.43 pA/pF after isoprenaline application. In addition, after isoprenaline application, a leftward shift of the current-voltage curve by approximately 6 mV was observed.

 

Fig. 3. Effects of isoprenaline on L-type calcium channels (ICa, L) of human ventricular cardiomyocytes: a, ICa, L recordings in human cardiomyocytes differentiated from induced pluripotent stem cells under control conditions (black trace) and after exposure to 1 µM isoprenaline (blue trace); b, current–voltage (I–V) relationships for ICa, L under control conditions (black trace, n = 6) and after isoprenaline (blue trace, n = 4); c, histogram of amplitude changes, percent under control conditions (black bar, n = 6) and after isoprenaline (blue bar, n = 4) (p ≤ 0.004). V indicates potential measured in millivolts (mV).

 

For IKs current recording, a stimulation protocol from −30 to +60 mV in 15 mV increments with a duration of 2.5 s was used. A representative example of such currents is shown in Fig. 4, a. Subsequently, current-voltage curves were plotted for control and isoprenaline conditions (Fig. 4, b). The current amplitude was normalized to cell capacitance, and at +60 mV, the maximum IKs current density in the protocol was 3.50 ± 0.49 pA/pF under control conditions and 6.94 ± 0.42 pA/pF in the presence of isoprenaline. Based on the histogram of normalized amplitudes expressed as percentages, the IKs current amplitude increased by approximately 98% after isoprenaline administration.

 

Fig. 4. Effects of isoprenaline on delayed rectifier potassium channels (IKs) of human ventricular cardiomyocytes: a, IKs currents in human cardiomyocytes differentiated from induced pluripotent stem cells under control conditions (black trace) and after exposure to 1 µM isoprenaline (blue trace); b, current–voltage (I–V) relationships for IKs under control conditions (black trace, n = 3) and after isoprenaline (blue trace, n = 3); c, histogram of amplitude changes, percent under control conditions (black bar, n = 3) and after isoprenaline (blue bar, n = 3) (p ≤ 0.008). V indicates potential measured in millivolts (mV).

 

Discussion

This study demonstrated a marked increase in voltage-gated channel currents in human cardiomyocytes under conditions of β-adrenergic stimulation.

One limitation of the study is that the degree of maturity of cardiomyocytes differentiated from the iPSC line may not be equivalent to that of adult human cardiomyocytes [20]. In the present work, cardiomyocytes obtained through directed differentiation exhibited AP morphology characteristic of ventricular-like cells, and AP duration also corresponded to that of ventricular cardiomyocytes. Based on our laboratory experience, the m34sk3 line reaches maximal electrophysiological maturity by day 30, as confirmed by stable conduction velocity values and response to periodic stimulation within the physiological range [21].

Another limitation is that currents were recorded at physiological temperature (37 °C). Measurement of INaV is technically challenging because of its large amplitude and rapid kinetics, particularly at physiological temperature. A separate study demonstrated that this may introduce systematic errors in the measurement of INaV activation and inactivation parameters, which can be accounted for by mathematical modeling of experimental artifacts [22]. It should be emphasized that the present study reports native current values without model-based correction, whereas further interpretation using mathematical modeling represents a separate task.

Modulation of ICa, L after the addition of isoprenaline increased the current amplitude of these channels by 120%, with a leftward shift of the peak of the current-voltage curve by 6 mV. Such an increase in ICa, L, together with other factors, leads to elevation of cytosolic calcium ion concentration, thereby enhancing myocardial contractility [13]. Reactivation of ICa, L increases the propensity for early afterdepolarizations, which are a major cause of fatal ventricular arrhythmias in long QT syndromes and heart failure [23].

The INaV current increased by approximately 73% under the influence of isoprenaline, with a 5-mV leftward shift of the activation curve. During β-adrenergic stimulation, protein kinase A–dependent phosphorylation enhances INaV by altering conductance [24]. This may contribute to sympathetically mediated acceleration of conduction velocity and to the development of recurrent arrhythmias after myocardial infarction, which are often characterized by myocardial depolarization [25].

After the addition of isoprenaline, IKs channel current amplitude increased by 98%. Under normal conditions, the density of the slow component of IKs potassium channels is lower than that of the rapid component IKr in humans and other large mammals [26]. However, β-adrenergic stimulation is known to increase IKs whereas it has almost no effect on IKr [27], thereby counteracting the increase in ICa, L and preventing prolongation of AP duration. Indeed, physical exertion and stress are typical triggers of arrhythmias in congenital long QT syndrome type 1, which is associated with loss of IKs function [28], and these can be prevented by β-adrenergic receptor blockade [29]. Computer modeling has demonstrated that an increase in the IKs/IKr ratio without prolongation of AP duration limits the occurrence of early afterdepolarizations. It is possible that IKs are more effective than IKr in stabilizing AP duration and suppressing early afterdepolarizations [30].

It should be noted that the difference in kinetics between the faster activation of ICa, L and the slower increase of IKs during β-adrenergic receptor activation transiently disrupts the balance of inward and outward currents [31]. This imbalance may transiently prolong AP duration and promote early afterdepolarizations, as demonstrated by computer modeling [32]. Moreover, the faster activation of ICa, L (compared with IKs) during rapid β-adrenergic receptor stimulation transiently increases AP restitution, leading to reentry breakdown and acceleration of the transition from ventricular tachycardia to ventricular fibrillation [33].

Conclusion

This study established that even at an isoprenaline concentration of 1 µM, there was a significant increase in the voltage-gated ionic currents INaV, ICa, L, and IKs in human cardiomyocytes derived from iPSCs. It was demonstrated that the difference in kinetics between the faster activation of ICa, L and the slower increase of IKs during β-adrenergic receptor activation transiently disrupts the balance of inward and outward currents, which may prolong AP duration and promote early afterdepolarizations. This mechanism may contribute to the development of life-threatening arrhythmias.

Additional information

Author contributions: S.G. Kovalenko: investigation (patch-clamp), formal analysis, writing—original draft; Sh.R. Frolova: conceptualization, investigation (patch-clamp), formal analysis, writing—original draft; S.A. Romanova: investigation (maintenance and differentiation of induced pluripotent stem cells); V.A. Tsvelaya: conceptualization, investigation (cell culture); R.A. Syunyaev: conceptualization, writing—original draft; K.I. Agladze: conceptualization, writing—original draft, writing—review & editing. All the authors approved the version of the manuscript to be published and agreed to be accountable for all aspects of the work, ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Acknowledgments: The authors express their gratitude to Veronika O. Abrasheva and the staff of the Laboratory of Molecular and Cellular Diagnostics at the Moscow Regional Research and Clinical Institute, as well as the staff of the Laboratory of Experimental and Cellular Medicine at the Moscow Institute of Physics and Technology.

Ethics approval: The study was conducted in accordance with the Helsinki Declaration (2024) and was approved by the Ethics Committee of the Moscow Regional Research and Clinical Institute (protocol No. 7 dated April 18, 2024) and the Interim Committee on Animal Care and Research Procedures of the Moscow Institute of Physics and Technology (protocol No. A2-2012-09-02 dated September 2, 2012). The cell line was provided by the National Medical Research Center named after academician E.N. Meshalkin. Treatment was approved by the Ethics Committee of the Institute of Circulatory Pathology (protocol No. 27 dated March 21, 2013). This induced pluripotent stem cell line was created using donated cells from a patient with cardiovascular disease. The authors obtained written informed consent from the donor.

Funding sources: The study was supported by the Ministry of Science and Higher Education of the Russian Federation (State Assignment No. FSMG-2023-0015, Agreement No. 075-03-2025-662, dated January 17, 2025), as well as by the Ministry of Health of the Moscow Region (State Assignment No. 61, M. F. Vladimirsky Moscow Regional Research Clinical Institute). The funding sources did not impose any restrictions on how the study results could be used or disseminated.

Disclosure of interests: The authors have no relationships, activities, or interests for the last three years related to for-profit or not-for-profit third parties whose interests may be affected by the content of the article.

Statement of originality: All figures in this work were created using original patch-clamp data.

Data availability statement: All data generated during this study are available in the article.

Generative AI: No generative artificial intelligence technologies were used to prepare this article.

Provenance and peer review: This paper was submitted unsolicited and reviewed following the standard procedure. The review process involved two members of the editorial board and the in-house scientific editor.

 

[1] The drug is not listed in the State Register of Medicinal Products of the Russian Federation.

[2] Szymanski MW, Singh DP. Isoproterenol. In: StatPearls. Treasure Island (FL): StatPearls Publishing; 2025 Jan–. Available at: http://www.ncbi.nlm.nih.gov/books/NBK526042/

About the authors

Sandaara G. Kovalenko

Moscow Regional Research and Clinical Institute; Moscow Institute of Physics and Technology

Email: sandaara.romanova@phystech.edu
ORCID iD: 0000-0003-3519-1962
SPIN-code: 8236-8387

Cand. Sci. (Physics and Mathematics)

Russian Federation, Moscow; Dolgoprudny

Sheida R. Frolova

Moscow Regional Research and Clinical Institute; Moscow Institute of Physics and Technology

Email: sh.frolova@monikiweb.ru
ORCID iD: 0000-0001-5325-8118
SPIN-code: 4604-2358

Cand. Sci. (Biology)

Russian Federation, Moscow; Dolgoprudny

Serafima А. Romanova

Moscow Regional Research and Clinical Institute; Moscow Institute of Physics and Technology

Email: scherbina_serafima@mail.ru
ORCID iD: 0000-0002-4223-0133
SPIN-code: 8562-6622
Russian Federation, Moscow; Dolgoprudny

Valeriya А. Tsvelaya

Moscow Regional Research and Clinical Institute; Moscow Institute of Physics and Technology

Email: vts@yandex.ru
ORCID iD: 0000-0002-3554-9736
SPIN-code: 7553-1038

Cand. Sci. (Biology)

Russian Federation, Moscow; Dolgoprudny

Roman A. Syunyayev

Email: roman.syunyaev@gmail.com
ORCID iD: 0000-0001-9582-0199
SPIN-code: 9735-7072

Cand. Sci. (Physics and Mathematics)

Russian Federation

Konstantin I. Agladze

Moscow Regional Research and Clinical Institute; Moscow Institute of Physics and Technology

Author for correspondence.
Email: agladze@yahoo.com
ORCID iD: 0000-0002-9258-436X
SPIN-code: 6960-8351

Dr. Sci. (Biology)

Russian Federation, Moscow; Dolgoprudny

References

Supplementary files