Synergistic antiarrhythmic mechanism of I_Na-L and RyR2 blockade: normalization of pathological calcium cycling and CaMKII signaling

Abstract

Background

Arrhythmias represent a leading cause of mortality among individuals with cardiovascular diseases. Considering the failure of antiarrhythmic drugs targeting single-ion channels in clinical trials, this study aims to evaluate the pharmacodynamics of various combinations of single-channel blockers to identify safer and more effective therapeutic regimens.

Methods

The antiarrhythmic effects of inhibitors alone and pairwise combination strategies targeting late sodium current (I_Na-L), hERG potassium current (I_Kr), ryanodine receptor 2 (RyR2), and L-type calcium current (I_Ca-L) inhibitors via a checkerboard dosing approach were evaluated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Comprehensive evaluation based on synergy scoring, efficacy, and toxicity (SynergyFinder) was performed to identify the preferred combination with high efficacy and low toxicity. The effectiveness of the preferred combination was further validated in isolated perfused guinea pig hearts. In addition, an analysis of the effects of the preferred combination on calcium sparks and Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was conducted.

Results

A combination regimen of the I_Na-L inhibitor GS967 and the RyR2 inhibitor Dantrolene, which exhibited highly effective, low-toxicity synergistic antiarrhythmic properties, was identified through checkerboard high-throughput screening. An alternative combination of the I_Na-L blocker Eleclazine and the RyR2 inhibitor TMDJ-035 showed similar therapeutic efficacy, indicating that the effectiveness of the preferred combination stems from target-mediated effects. The combination further demonstrated significant efficacy in multiple hiPSC-CM arrhythmia models and an ex vivo guinea pig heart reperfusion arrhythmia model. Mechanistic studies revealed that the dual-target inhibition strategy corrected pathological calcium sparks and calcium cycling dysfunction, downregulated the CaMKII-RyR2 phosphorylation cascade, thereby restoring electrical signaling.

Conclusions

The combination of I_Na-L and RyR2 inhibitors exerts safer and more effective antiarrhythmic effects. This combinational therapeutic strategy provides a theoretical basis for developing novel antiarrhythmic regimens based on dual-target regulation, demonstrating significant translational medical potential and warranting further clinical evaluation.

Graphical Abstract

Supplementary Information

The online version contains supplementary material available at 10.1186/s12967-025-07652-3.

Background

Arrhythmias arise from disturbances in the electrical activity of the heart, among which, rapid ventricular arrhythmias such as ventricular fibrillation (VF) can lead to sudden cardiac death (SCD), constituting one of the primary causes of mortality in patients with cardiovascular diseases [1]. Despite significant advancements in interventional therapies, including device-based treatments and cardioneuroablation [2], pharmacotherapy remains the first-line treatment option for the majority of clinical patients [3]. The Vaughan Williams classification scheme categorizes antiarrhythmic drugs into four classes, including sodium channel blockers, beta receptor blockers, action potential duration (APD) prolonging agents, and calcium channel blockers. The modern classification of antiarrhythmic drugs proposed in 2018 has included late sodium current (I_Na-L) and ryanodine receptor type 2 (RyR2) inhibitors, and it remains dominated by drugs that are blockers of various ion channels [4, 5]. However, ion channel blockers often prove ineffective in clinical trials and may even increase mortality rates [6, 7]. Hence, there is a pressing demand for the development of novel drugs or therapeutic strategies.

The electrical activity of the heart depends on the precise regulation of transmembrane ion homeostasis, in which the action potential (AP) and calcium cycling constitute the core regulatory hubs [8]. The inward currents mediated by Na⁺ and Ca²⁺ channels on the cardiomyocyte membrane dominate the depolarization of AP. The outward currents mediated by various K⁺ channels are responsible for the repolarization of AP [9]. RyR2, as an intracellular Ca²⁺ release channel, triggers calcium-induced calcium release (CICR) through the influx of Ca²⁺ introduced by the opening of L-type calcium channels, driving myocardial contraction [10]. In turn, intracellular Ca²⁺ feedback regulates the AP profile through signaling pathways such as Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) and Na⁺/Ca²⁺ exchangers (NCX). The bidirectional coupling of Ca²⁺ and ion channels on the cardiomyocyte membrane jointly regulates AP generation and conduction [11]. Dysfunction at any stage may lead to arrhythmias.

Multiple studies indicate that the pathogenesis of arrhythmias typically involves complex abnormalities in multiple ion channels rather than dysfunction of a single channel [12–15]. Antiarrhythmic drugs targeting only a single ion channel often yield limited efficacy, suggesting that modulating the balance of multiple channels in myocardial electrophysiology may enhance effectiveness. Conversely, excessive inhibition of a specific ion channel may disrupt ion current homeostasis and thus pose a risk of proarrhythmia. An example is hERG potassium channel blockade, which prolongs APD and makes the heart prone to torsades de pointes arrhythmias (TdP) [16]. Studies have indicated that I_Na-L inhibitors effectively reduce the risk of torsade de pointes caused by hERG block, demonstrating that multi-channel regulation strategies may mitigate hazards [17]. The above studies indicate that combining drugs targeting different channels may exert highly efficient and low toxicity antiarrhythmic effects. However, there is currently a lack of pharmacological evaluation on the combined use of different ion channel blockers. Therefore, the purpose of this study was to evaluate the pharmacodynamics of combinations of ion channel targets, including late sodium current (I_Na-L), hERG potassium current (I_Kr), L-type calcium current (I_Ca-L), and ryanodine receptor 2 (RyR2) inhibitors, and identify safer and more effective antiarrhythmic therapies. This represents the first systematic evaluation of multi-target combination strategies, with drug efficacy quantified through multi-model collaborative scoring.

Methods

Experimental animals

Adult male Dunkin-Hartley guinea pigs (250 ~ 350 g) were purchased from the Beijing Changyang Xishan breeding farm. During the rearing period, conditions were kept at 20–30 °C, with 60% humidity and a 12:12 light-dark cycle. Before the experimental procedures, the animals were allowed a week to become accustomed to the new environment, and the health status of the guinea pigs was observed daily by the researchers. All animal experimental procedures were approved by the Animal Welfare Committee of Hebei Medical University (Shijiazhuang, China) and conducted in accordance with the guidelines published by the National Institutes of Health for the Care and Use of Laboratory Animals (publication No. 85 − 23, revised 1996). (approval-number-Hebmu-2023046, Shijiazhuang, China).

Differentiation, culture, and reinoculation of hiPSC-CMs

The human induced pluripotent stem cells (hiPSCs) provided by Cellapy were derived from continuous cultured in the PGM1 culture system to reach a steady state (CA1007500; Cellapy, Beijing, China). Furthermore, these hiPSC clones, between passages 20 and 30, were differentiated into ventricular cardiomyocytes via the cardioeasy chemically defined cardiac differentiation kit (CA2004500; Cellapy, Beijing, China). At 90%-95% hiPSCs confluence, medium was switched to CardioEasy^® Differentiation Medium I to initiate differentiation. After 48 h, sequentially switch to Differentiation Medium II and III for induction. Spontaneously contracting cell clusters become observable on days 8 ~ 10. On days 13 ~ 16, the cells were purified using cardiomyocyte purification medium, with the maintenance medium adjusted based on the basis of cellular contractility. Before each medium was changed, the cells were washed with calcium - and magnesium-free DPBS. After the cell stabilization, the cells were digested and reinoculated.

Electrophysiological recording of hiPSC-CMs

The differentiated hiPSC-CMs were then seeded onto MEA-96-well electrode plates at a density of 5 ~ 8 × 10⁴ cells/well. After 5 ~ 7 days of culture to achieve a synchronized monolayer, the cells underwent electrophysiological signal detection, including field potential, contraction, and conduction, via the Mestro multi-well microelectrode array (MEA) platform. Alternatively, programmed electrical stimulation (PES) was used to induce arrhythmias [18].

Checkerboard dosing and evaluation

A six-level arrhythmia scoring system was established to quantify efficacy [19, 20]. Normal electrical activity (Grade 0), EAD (Grade 1), rolling EAD (Grade 2), ectopic beats (Grade 3), irregular beating (Grade 4), tachyarrhythmia or no beating (Grade 5). Randomized across plates to avoid positional bias. SynergyFinder uses cNMF algorithm to detect and replace outlier measurements [21]. Arrhythmogenic effects under therapy are defined as toxic reactions. In the hiPSC-CM arrhythmia model, different channel-selective blockers or their checkerboard combinations were added. Arrhythmia-like electrical activity was observed and quantified according to established grading criteria. The median effect concentration (EC₅₀) is calculated [22]. The intervention effects of the combinations were analyzed for synergistic interactions via the SynergyFinder Plus platform [23], with the mean percentage of cellular arrhythmia suppression and corresponding concentrations used as inputs. The Combined Sensitivity Score (CSS) was computed as described in Malyutina et al. [24], integrating synergy across all dose slices. The efficacy and toxicity of combination therapies were evaluated simultaneously through a multi-model synergistic evaluation system incorporating the Loewe Addition, Bliss, HSA, and ZIP models, thereby providing a holistic assessment of combination strategies. A score > 5 indicates synergy (red), < 0 indicates antagonism (blue), and = 0 indicates additivity (white).

Ex vivo electrocardiogram monitoring

After intraperitoneal injection of heparin sodium (1000 U/ml) for 15 min, the guinea pigs were anesthetized with isoflurane. The thoracic cavity was opened, the heart was quickly excised, the aorta was located, and excess tissue was removed. The aorta was promptly connected to a cannula and ligated with surgical sutures. Residual blood within the heart was expelled, and the heart was connected to the perfusion apparatus and stabilized for 15 min to restore normal heart rhythm. A 15-minute normal electrocardiogram was recorded. Record the electrocardiogram 15 min after the agents cycle. The preparation of the external solution for isolated heart perfusion was as follows, 132 mM NaCl, 5.4 mM KCl, 10 mM HEPES, 1 mM MgCl₂, 10 mM Glucose, and 2 mM CaCl₂, adjusted to pH 7.4 with NaOH. Retrograde perfusion through the aorta was performed via a peristaltic pump with freshly prepared and oxygenated Tyrode’s solution for 0.5 h (7–8 mL/min; 37 ± 0.5 °C).

Western blot

Protein levels were analyzed by Western blot. Whole-cell proteins were extracted from hiPSC-CMs. Prepare cell lysates using RIPA lysis buffer, with a protein loading of 20–30 µg. Separate equal amounts of proteins by SDS-PAGE and transfer them onto a polyvinylidene difluoride (PVDF) membrane (Sigma, USA). Block the PVDF membrane with 5% skim milk and incubate it overnight at 4 °C with primary antibodies. Next, incubate with goat anti-rabbit or anti-mouse secondary antibodies (Rockland, USA), at room temperature for two hours, then scan using the Odyssey Infrared Imaging System (LI-COR). Odyssey Imager software (version 3.0) was employed to detect the integrated intensity of the proteins. The change in protein content reflects the effects of the agents on functional channel proteins in the cells. The following primary antibodies were used: RyR2 (Immunoway, USA), p-RyR2-S2814 (Affinity, USA), p-CaMKII-T286 (Cell Signaling Technology, USA), CaMKII (Abcam, UK), and GAPDH (Proteintech Group, China).

Calcium imaging

The hiPSC-CMs were seeded and cultured in a 96-well plate with a transparent bottom and subjected to modeling and intervention. Following the removal of the culture medium, the hiPSC-CMs underwent three washes with D-Hank’s balanced salt Solution (HBSS). The cells were subsequently incubated with HBSS solution containing 5 µM Fluo4-AM and 0.02% F127 at 37 °C in the dark for 15 min. The HBSS was then replaced, and the environmental temperature of the high-content screening system (Molecular Devices, USA) was set to 37 °C to record spontaneous calcium transients and calcium sparks in the cells. ImageJ was used to convert the images into digital data.

Agent preparation

ET-1 was prepared as a 100 µM stock solution using triple-distilled water. Lidocaine, E-4031, Verapamil, and Nimodipine (50 mM each), as well as GS967, Dantrolene and Nifekalant (10 mM each), and ATX-II (20 µM) were prepared in DMSO. Stock solutions were stored at -20 °C and freshly diluted to working concentrations prior to each experiment.

Statistical analysis

In this study, AcqKnowledge 4.2.1, GraphPad Prism 10, and Odyssey were employed for image processing and statistical data analysis. Experimental results are expressed as the mean ± SEM, and the normality of the numerical data was assessed via the Shapiro-Wilk or Kolmogorov-Smirnov tests. Categorical data were analyzed via the χ² test. For comparisons between two groups, Student’s t-test was used for normally distributed data, whereas non-parametric tests (Mann-Whitney U) were applied for non-normally distributed data. For comparisons among multiple groups, for data that meet the assumptions of normality and homogeneity of variance, One-way ANOVA - Tukey’s post hoc test or One-way ANOVA - Dunnett’s test is used. Welch’s ANOVA test was applied for data that followed a normal distribution but exhibited unequal variances. The Kruskal-Wallis test was employed for data that did not meet the normality assumption.

Results

Pharmacological analysis of channel blockers alone in hiPSC-CMs

Through programmed induction, we successfully differentiated human-derived induced pluripotent stem cells (hiPSCs) into functional cardiomyocytes (hiPSC-CMs) (Fig. 1A). These cells highly expressed ventricular markers (Fig. S1A) and exhibited responses to classic ion channel blockers consistent with those of human cardiomyocytes (Fig. S1B-C), validating the reliability of the model [25, 26]. To simulate the most common ventricular arrhythmias secondary to pathological myocardial hypertrophy in clinical settings, we stimulated hiPSC-CMs with ET-1 for 24 h (Fig. 1A). The cells exhibited electrophysiological abnormalities, structural hypertrophy, and arrhythmia phenotypes (Fig. S1D-G), indicating the successful establishment of an arrhythmia model. To evaluate the antiarrhythmic effects of single-channel inhibitors, five selective ion channel blockers, namely, the I_Na-L inhibitor GS967, the hERG channel blockers E-4031 and Nifekalant, the L-type calcium channel blocker Nimodipine, and the RyR2 inhibitor Dantrolene were applied at increasing concentrations. Given the high arrhythmia risk associated with the classic hERG blocker E-4031 [27, 28], the safer pure potassium channel blocker Nifekalant was incorporated into this study. At concentrations less than 1 µM, Nifekalant specifically targets hERG channels [29].

Fig. 1.

Effects of inhibitors alone on ET-1-induced arrhythmias in hiPSC-CMs. (A) Schematic diagram of the controlled initiation of the differentiation process and experimental protocol for hiPSC-CMs. (B) Typical FP waveforms after interventions with different concentrations of GS967, Nifekalant, Nimodipine, Dantrolene, and E-4031 in the hiPSC arrhythmia model. (C) Statistical graph of the EC₅₀ values for agents used for arrhythmia intervention. n ≥ 3 per group

After 24 h of induced arrhythmia, the cells were treated with each compound (Fig. 1A). The field potential (FP) waveforms recorded by MEA after intervention are shown in the Fig. 1B. Except that the I_Na-L inhibitor GS967 showed only antiarrhythmic effects at the test concentrations, the other three target inhibitors exhibited dose-dependent bidirectional pharmacological effects, that is, antiarrhythmic effects at low concentrations and proarrhythmic effects at high concentrations. The calcium channel blocker Nimodipine induced cell arrest at high concentrations, whereas the hERG channel blockers Nifekalant and E-4031, at high concentrations, caused EAD, rolling EAD, and even ventricular tachycardia-like arrhythmias. The RyR2 inhibitor Dantrolene at high concentrations also induced irregular beating. Statistical analysis revealed that the median effect concentration (EC₅₀) values for the antiarrhythmic effects were 53.08 nM for GS967, 463.3 nM for Nifekalant, 1.52 nM for E-4031, 0.76 nM for Nimodipine, and 158.9 nM for Dantrolene, respectively (Fig. 1C). The above results suggest that most single-channel inhibitors alone generally carry the risk of proarrhythmia and have a narrow therapeutic window, whereas I_Na-L inhibitor has relatively good safety profiles.

Pharmacodynamic quantitative evaluation of the combined channel blockers

Further pairing of the four classes of channel blockers resulted in six antiarrhythmic therapeutic strategies, namely, GS967-E-4031, GS967-Nifekalant, GS967-Nimodipine, GS967-Dantrolene, Nifekalant-Nimodipine, and Nifekalant-Dantrolene. Due to the lack of a definitive synergy model to measure the intricate synergistic interactions between agents [30], we utilized various synergy quantification metrics to thoroughly assess potential synergistic interactions. We performed high-throughput screening of combinations via a checkerboard assay in hiPSC-CMs and visualized the combination effects with the SynergyFinder tool for synergistic quantification [31], as exemplified by the Loewe model (Fig. 2A).

Fig. 2.

Comprehensive evaluation of combinations. (A) The combined effects of the six combinations were visualized via SynergyFinder software and scored via the Loewe model. (B) Heatmap for assessing the scores of multiple synergy models. (C) Statistical plots of combination synergy scores and combined efficacy and toxicity (proarrhythmic risk) evaluations. All experiments were performed with at least three independent replicates

Unlike E-4031 alone, which induced EAD, rolling EAD and even ventricular tachycardia-like electrical activity at high concentrations (Fig. 1B), the combination of GS967 and E-4031 partially counteracted the abnormal electrical activities induced by E-4031. Except for the ZIP model, the Loewe, Bliss, and HSA models all presented certain synergistic regions (Fig. S2A-B).

Similarly, combined treatment with GS967 partially alleviated the electrical activity abnormalities induced by Nifekalant. The Loewe, Bliss, HSA, and ZIP models all show certain synergistic regions (Fig. S3A-B). The results indicated that GS967 partially antagonized the electrical activity abnormalities induced by high concentrations of E-4031 and Nifekalant, and that combination therapy may reduce the arrhythmogenic risk of hERG inhibitors.

Nimodipine alone exhibited significant cytotoxicity at high concentrations, easily inducing cell arrest (Fig. 1B). GS967 is unable to reverse the electrical quiescence phenomenon. No synergistic regions are observed in the Loewe, Bliss, HSA, and ZIP models (Fig. S4A-B), indicating low synergy between the two agents.

High concentrations of Dantrolene alone were prone to induce irregular cellular contractions (Fig. 1B), whereas GS967 mitigated the risk of irregular contractions caused by Dantrolene. The combination completely suppressed abnormal electrical activity in certain regions, achieving highly effective antiarrhythmic effects at low dose ratios (e.g., 100 nM GS967-100 nM Dantrolene). The Loewe, Bliss, HSA, and ZIP models all exhibited extensive synergistic regions (Fig. S5A-B).

When Nifekalant was combined with Nimodipine, it was unable to reverse the electrical quiescence caused by Nimodipine. Although the ZIP and Bliss models show small synergistic regions, there are no synergistic regions in the Loewe and HSA models (Fig. S6A-B).

The combination of Nifekalant and Dantrolene increased the occurrence of EAD or rolling EAD. Only the Loewe model shows a certain synergistic region (Fig. S7A-B).

A heatmap illustrating the comparison of scores from the Loewe, Bliss, HSA, and ZIP models was used to evaluate the efficacy of the six inhibitor combination strategies comprehensively. In the Loewe, Bliss, HSA, and ZIP models, a higher synergy score indicates stronger synergistic effects, with deeper red representing the strongest synergy. The combination of GS967-Dantrolene meets all the synergistic criteria of the reference models, followed by GS967-Nifekalant and Nifekalant-Dantrolene, with the Loewe model presenting an additive effect. The combinations of GS967-E-4031, GS967-Nimodipine, and Nifekalant-Nimodipine are predominantly characterized by antagonistic effects (Fig. 2A-B).

The combined sensitivity score (CSS) can serve as a quantitative measure of therapeutic efficacy to determine the sensitivity of the agent pairs [32], with the GS967-Dantrolene score remaining the highest, the GS967-Nifekalant score comes next, followed sequentially by Nifekalant-Dantrolene, GS967-E-4031, Nifekalant-Nimodipine and GS967-Nimodipine combination scores are less than zero. Further toxicity evaluation revealed that GS967-Dantrolene and Nifekalant-Dantrolene exhibited the lowest toxicity, followed by GS967-Nifekalant, GS967-E-4031, Nifekalant-Nimodipine, and GS967-Nimodipine. On the basis of the synergistic model scoring, efficacy, and comprehensive toxicity evaluation system, the combination of I_Na-L and RyR2 inhibitors (GS967-Dantrolene) was ultimately identified as the preferred combination (Fig. 2C).

Target-specific effects of synergistic anti-arrhythmic effects

To verify whether the synergistic antiarrhythmic effects of GS967-Dantrolene stem from their specific actions on targets (I_Na-L and RyR2) rather than off-target effects, we further performed a pharmacodynamics study using the alternative I_Na-L inhibitor, Eleclazine (GS-6615), and the RyR2 inhibitor, TMDJ-035. The FP waveforms after intervention are shown in the Fig. 3A. Neither Eleclazine nor TMDJ-035 alone significantly affected FPDc, but their combined application produced a significant inhibitory effect, maintaining FPDc at pre-intervention levels (Fig. 3B). In the electrical stimulation (PES)-induced arrhythmia, combination therapy exhibited significant resistance, whereas the monotherapy group remained highly susceptible (Fig. 3C). The above results indicate that Eleclazine and TMDJ-035 individually do not exhibit significant antiarrhythmic effects, whereas their combination significantly suppresses spontaneous arrhythmias and counteracts PES-induced arrhythmias. The synergistic antiarrhythmic action demonstrates the cooperative potential of dual blockade of I_Na-L and RyR2 channels, thereby providing substantial support for the rationale and feasibility of the dual-target combination strategy.

Fig. 3.

Therapeutic effects of Eleclazine and TMDJ-035 on ET-1-induced arrhythmia in hiPSC-CMs. (A) Typical schematic of the FP of hiPSC-CMs treated with Eleclazine and TMDJ-035. (B) Statistical plot of FPDc after modeling and intervention. One-way ANOVA -Tukey test. n ≥ 3 per group. (C) Incidence of PES-induced arrhythmias. χ² test. n ≥ 22 per group. *P < 0.05 vs Before, #P < 0.05 vs. ET-1, &P < 0.05 vs Ele-TMDJ

Pharmacological evaluation of the preferred combination in multiple arrhythmic models of hiPSC-CMs

Subsequently, we evaluated the effectiveness of the preferred combination in arrhythmia models induced by alternative stimuli since arrhythmias are clinically induced by different etiologies. Similar to ET-1 (200 nM/1 µM), Angiotensin II (Ang II, 200 nM/1 µM), Phenylephrine (PE, 10/20/50 µM), and H₂O₂ (5 µM) increased the cell area over time, with effects stabilizing after 24 h of challenge (Fig. 4A-B). Field potential signals revealed that Ang II (200 nM), PE (10 µM), and H₂O₂ (5 µM) all induced arrhythmic phenotypes in hiPSC-CMs. The preferred combination mitigated the arrhythmias induced by these agents (Fig. 4C). Furthermore, this combination therapy was validated in a hiPSC-CM hypoxia-reoxygenation model. Under pathological conditions, GS967-Dantrolene most effectively enhanced cell survival and reduced reactive oxygen species (ROS) production (Fig. S8A-C). These findings indicate the therapeutic potential of the dual-target strategy for suppressing abnormal electrical activity and suppressing damage triggered by diverse etiologies.

Fig. 4.

Protective effects of the preferred combination on cellular and isolated heart model. Typical schematic (A) and statistical graph (B) demonstrating that CTL, ET-1, Ang II, PE, and H₂O₂ modeling resulted in an increase in the cell area of hiPSC-CMs with prolonged intervention. (C) Schematic diagram of typical FP waveforms for arrhythmia modeling and preferred combination interventions. (D) Typical ECG tracings of ischemia-reperfusion-induced arrhythmias and pharmacologic interventions in isolated guinea pig hearts. (E) Statistical plot of the incidence of VT/VF. χ² test. (F) Statistical plot of the duration of VT/VF. Kruskal-Wallis test. (G) Statistical chart of the arrhythmia scores. Kruskal-Wallis test. n ≥ 7 per group. * P < 0.05, ** P < 0.01, ***P < 0.001 vs untreated

Antiarrhythmic effects of the preferred combination in isolated perfused guinea pig hearts

The pharmacological effects of four classes of channel blockers were subsequently assessed in an arrhythmia model induced by ischemia-reperfusion in isolated perfused guinea-pig hearts. Initially, agents alone or in combination were preperfused for 15 min, followed by ischemia for 30 min and reperfusion to induce arrhythmias. Figure 4D shows the electrocardiogram (ECG) after the restoration of heartbeat following cardiac reperfusion. All untreated hearts developed severe ventricular arrhythmias, predominantly ventricular fibrillation (8/9; duration 90.18 ± 14.88 s), fully validating the stability and reliability of this model. Both monotherapy and various combinations significantly reduced the incidence and duration of VT/VF. Among all combinations, the incidence of VT/VF differed little between GS967-E4301 and GS967-Dantrolene (the preferred combination), followed by GS967-Nimodipine (Fig. 4E). The S967-Dantrolene combination resulted in the shortest duration of VT/VF following intervention (Fig. 4F). Further quantitative analysis of arrhythmia severity via the Curtis and Walker (1988) scoring system [33] revealed that both monotherapy and various combinations significantly reduced arrhythmia scores, with the GS967-Dantrolene combination yielding the lowest scores. The preferred combination exhibited superior potency compared with the same dose of low-concentration monotherapy, exhibiting a synergistic interaction (Fig. 4G). These findings confirm that at the organ level, the occurrence and progression of ischemia-reperfusion-induced arrhythmias are closely associated with a vicious cycle involving increased I_Na-L and intracellular calcium overload. Simultaneous targeting of these two key pathological pathways through synergistic blockade produces potent antiarrhythmic effects.

Effect of the preferred combination on intracellular calcium homeostasis

After confirming the superior efficacy of the preferred combination for the prevention and treatment of arrhythmias, we further explored its mechanism of action. The preferred combination intervention reversed electrical abnormalities and contractile dysfunction while eliminating reentry phenomena (Fig. 5A). Statistical analysis revealed that the preferred combination significantly shortened FPDc (Fig. 5B) and reduced beat period irregularity (Fig. 5C), although it failed to fully restore the conduction velocity to baseline levels (Fig. 5D). The presence of contractile dysfunction suggests that disrupted calcium signaling may represent the core pathological mechanism underlying arrhythmia induction, and the dual-target strategy may exert its therapeutic effects by modulating calcium handling and electrical coupling processes.

Fig. 5.

Preferred combination reversed calcium signaling dysfunction in hiPSC-CMs. (A) Typical plots of FP, AP, contractile function, and conduction signals of hiPSC-CMs in the control group, model group, and preferred combination intervention group. (B-D) Statistical plots of FPDc (One-way ANOVA -Tukey test), beat period irregularity (Kruskal-Wallis test), and conduction velocity (Kruskal-Wallis test). n ≥ 7 per group. (E-F) Typical and statistical plots of the effects of GS967-Dantrolene alone or in combination on reducing the number of spontaneous calcium transient Ca²⁺ release events induced in hiPSC-CMs. Kruskal-Wallis test. n ≥ 19 per group. (G-H) Typical and statistical plots of the ability of GS967-Dantrolene alone or in combination to reverse the increase in the calcium spark frequency (CaSpF) in quiescent hiPSC-CMs. Kruskal-Wallis test. n ≥ 10 per group. *P < 0.05, ** P < 0.01, *** P < 0.001 vs CTL. #P < 0.05, ## P < 0.01 vs ET-1

To investigate changes in intracellular calcium signaling processing, we analyzed hiPSC-CMs via calcium imaging technology. Compared with the CTL group, the model group exhibited varying degrees of calcium homeostasis disruption (Fig. S9). During spontaneous beating, spontaneous Ca²⁺ release events in hiPSC-CMs from the model group were interspersed between Ca²⁺ transients, which were rebalanced following the preferred combination intervention (Fig. 5E). Quantitative analysis revealed that the frequency of spontaneous calcium release events in the model group was significantly greater than that in the CTL group. Neither GS967 nor Dantrolene alone significantly affected this frequency, but their combined application had a significant effect (Fig. 5F). Additionally, the calcium spark frequency (CaSpF) in the model module significantly increased. Neither agent alone significantly improved CaSpF, but the combination therapy effectively reversed this phenomenon, causing a marked decrease in CaSpF (Fig. 5G-H). In summary, these results indicate that the preferred combination rescues arrhythmias by effectively correcting abnormal Ca²⁺ handling in hiPSC-CMs.

Effect of the preferred combination on CaMKII/RyR2 phosphorylation cascade

In addition to direct modulation of ion channels, the synergistic antiarrhythmic effects of preferred combination may stem from their shared regulation of upstream signaling pathways. RyR2-mediated diastolic sarcoplasmic reticulum (SR) Ca²⁺ leakage is often associated with channel phosphorylation [34]. CaMKII activation can simultaneously increase the phosphorylation of RyR2 and Na_V1.5 channels, leading to increased Ca²⁺ leakage and increased I_Na-L [35–37], jointly contributing to an arrhythmogenic substrate. On the basis of these findings, we hypothesize that the preferred combination may exert synergistic effects through the CaMKII pathway.

To validate this hypothesis, we detected key proteins in the CaMKII signaling pathway via Western blot analysis (Fig. 6A). The results revealed that the expression level of the P-RyR2-S2814 in the modeling group was significantly upregulated. Neither GS967 nor Dantrolene alone had a significant effect on P-RyR2-S2814 expression levels. Following combined intervention, P-RyR2-S2814 expression levels were significantly reduced (Fig. 6B). Neither modeling nor intervention affected the expression of the RyR2 protein (Fig. 6C). Modeling also induced significant upregulation of CaMKII and its phosphorylation at T286. Following GS967 intervention, P-CaMKII-T286 expression levels decreased significantly, whereas Dantrolene intervention had no significant effect. Combined treatment with GS967 and Dantrolene intervention resulted in a significant reduction in P-CaMKII-T286 expression (Fig. 6D). Neither GS967 nor Dantrolene alone significantly affected CaMKII expression levels; however, combined intervention significantly reduced CaMKII expression (Fig. 6E). GS967 and Dantrolene reversed the upregulation of CaMKII, P-CaMKII-T286, and P-RyR2-S2814 protein expression. Compared with either agent alone, the combined therapy had stronger inhibitory effects on key molecules in the CaMKII pathway, suggesting that GS967 and Dantrolene synergistically restore myocardial electrical stability by inhibiting the CaMKII/RyR2 phosphorylation cascade.

Fig. 6.

Preferred combination treated electrophysiological disorders in hiPSC-CMs by inhibiting the activated CaMKII pathway. Western blot (A) and quantitative analysis revealed the effects of GS967-Dantrolene alone and in combination therapy on the expression of P-RyR2-S2814 (B), RyR2 (C), P-CaMKII-T286 (D), and CaMKII (E) in hiPSC-CMs. n = 3 per group. One-way ANOVA- Dunnett test. *P < 0.05, ** P < 0.01, *** P < 0.001 vs CTL. #P < 0.05, ## P < 0.01 vs ET-1

To further clarify the central role of CaMKII in arrhythmias, we employed its inhibitor, KN-93, for validation. The inhibitor KN-93, frequently utilized for investigating the cellular and in vivo functions of CaMKII, reduces CaMKII activity through its interaction with Ca²⁺/CaM [38]. MEA recordings revealed that KN-93 treatment significantly alleviated model-induced electrophysiological disturbances, eliminating phenomena such as EADs, rolling EADs, delayed afterdepolarization (DAD), and irregular beats (Fig. S10A). Statistical analysis revealed that KN-93 reversed the prolongation of the FPDc and increased beat period irregularity induced by modeling (Fig. S10B-C), suggesting that the synergistic antiarrhythmic effect of the combination therapy was achieved by inhibiting excessive CaMKII activation. In summary, GS967 and Dantrolene exert synergistic antiarrhythmic effects by jointly inhibiting the CaMKII/RyR2 phosphorylation cascade, thereby correcting abnormal calcium handling, impaired contractile function, and electrophysiological disturbances.

Discussion

In this study, we proposed and evaluated an antiarrhythmic strategy that involves dual inhibition of I_Na-L and RyR2 through quantitative pharmacodynamics. This combined approach demonstrates both safety and efficacy, successfully avoiding toxicity concerns associated with single-channel blockers while exhibiting potent synergistic antiarrhythmic effects at low concentrations. Furthermore, this strategy improves calcium homeostasis imbalance and electrophysiological disturbances by regulating the CaMKII/RyR2 phosphorylation cascade, thereby demonstrating antiarrhythmic efficacy across multiple pathological models.

Traditional single-target antiarrhythmic drugs face clinical limitations due to the “therapeutic window dilemma”. In contrast, combination therapy has been widely adopted for treating tuberculosis, hypertension, viral myocarditis and many drug-resistant cancers such as gliomas [31–40]. Rational combination therapy can produce synergistic effects where “1 + 1 > 2”, helping to reduce single-agent dosages, minimize side effects, and delay the onset of drug resistance [41]. However, the potential for antagonistic effects also exists. The synergistic interactions among antiarrhythmic drugs have not been fully characterized or tested in appropriate disease models. Specifically, data on effective dosages and drug interactions remain scarce, necessitating further investigation. Additionally, the pharmacological effects of combination therapy require quantitative evaluation via diverse computational models. In the absence of a gold-standard synergy model to quantify complex drug interactions, this study employs multiple synergy metrics to comprehensively assess potential synergistic effects among drug combinations.

The most common clinical ventricular arrhythmias typically arise from pathological myocardial hypertrophy induced by chronic pathological stressors such as hypertension, myocardial infarction, or valvular dysfunction [42]. During the compensatory phase, these patients can maintain stable cardiac function [43, 44]; however, persistent pathological stimuli gradually overwhelm compensatory mechanisms. Patients then face not only the risk of persistent ventricular arrhythmias but also accelerated heart failure progression due to impaired electromechanical coupling [45], underscoring the urgent need for early intervention strategies. Traditional whole-animal or isolated heart arrhythmia models cannot fully replicate the electrophysiological characteristics of human cardiomyocytes and struggle to meet the high-throughput quantitative efficacy assessment demands for drug combinations. The emergence of hiPSC-CMs in recent years has broken through clinical research bottlenecks, providing a cellular model capable of simulating pathological arrhythmias and enabling high-throughput drug screening and efficacy observation. This study confirmed that hERG, L-type calcium channel, and RyR2 inhibitors exhibit proarrhythmic effects at high concentrations due to excessive disruption of ion homeostasis, such as E-4031, exhibit proarrhythmic effects at high concentrations due to excessive disruption of ion homeostasis, which is consistent with the clinically observed risk of quinidine-induced TdP [46]. I_Na-L inhibitors have safety advantages, yet single-target interventions remain insufficient to fully interrupt the vicious cycle of arrhythmias. The screened dual-target combined inhibition strategy targeting both I_Na-L and RyR2 exhibited favorable safety and efficacy and was validated across multiple arrhythmia models.

Electrophysiological and structural biology studies have established I_Na-L and RyR2 as critical regulatory nodes in the onset and progression of cardiac arrhythmias [47–49]. The I_Na-L represents a small, sustained inward sodium current generated by the incomplete inactivation of voltage-gated sodium channels (primarily Na_V1.5) during the action potential plateau phase. Under physiological conditions, its amplitude is minimal, accounting for approximately 0.5% of the peak I_Na, yet it plays a vital role in maintaining the plateau phase. Under conditions of genetic mutation [50] or pathological states [15, 51, 52], the I_Na-L can increase to 4%-5% of the peak sodium current. Notably, even a modest increase of 0.3%-1% in the I_Na-L is sufficient to significantly elevate the risk of arrhythmia. In this study, we observed a significant prolongation of the FPDc following ATX-II-induced I_Na-L enhancement, which was effectively reversed by GS967 (Fig. S11). On the other hand, RyR2, as the calcium release channel on the sarcoplasmic reticulum, is central to the excitation-contraction coupling. RyR2 dysfunction—induced by hyperphosphorylation [53–55], oxidative stress, or dissociation of stabilizing subunits (e.g., FKBP12.6 [56, 57]) can cause diastolic calcium leakage, triggering DAD as another major arrhythmia mechanism. Enhanced I_Na-L and RyR2-mediated calcium leakage frequently coexist in various pathological arrhythmias [13, 58]. Our experimental results demonstrate that a dual-target combined inhibition strategy has synergistic effects across multiple pathological models, eliminating the risks associated with high-dose monotherapy. This synergy is not attributable to off-target effects but is confirmed to originate from specific interactions between the two targets. In conclusion, dual-target combined inhibition represents a promising novel therapeutic strategy for antiarrhythmic effects.

In this study, treatment with the CaMKII inhibitor KN-93 alone significantly improved FPDc and rhythm stability, suggesting that excessive CaMKII activation is a core molecular event linking calcium homeostasis to electrophysiological abnormalities. Previous evidence has demonstrated that activated CaMKII can directly phosphorylate both the Na_V1.5 channel and RyR2 [36, 37, 59, 60]. On the basis of the experimental data, we propose the following mechanism pathway, wherein during arrhythmia states, CaMKII activates and phosphorylates the Na_V1.5 channel, delaying its inactivation and leading to increased I_Na-L, prolonging APD, and increasing repolarization dispersion, thereby inducing EAD. Concurrently, CaMKII-mediated phosphorylation of RyR2 at site S2814 elevates the probability of channel opening, triggering abnormal calcium release from the SR and causing intracellular calcium overload. This overload further activates NCX, generating a net inward current and inducing membrane depolarization, thereby forming DAD. Disrupted calcium signaling significantly impairs cellular contraction function. Abnormal calcium release further exacerbates calcium homeostasis disruption by activating CaMKII, creating a vicious cycle [36, 61]. Dual-targeted combined intervention simultaneously addresses these two critical points, effectively breaking the vicious cycle and correcting abnormal electrical activity (Fig. 7). These findings provide novel experimental evidence and therapeutic insights for preventing and treating arrhythmias in complex pathological contexts.

Fig. 7.

Mechanism diagram. In arrhythmia models, CaMKII activation leads to the phosphorylation of Na_V1.5 channels, resulting in delayed sodium channel inactivation, the generation of persistent late sodium current, prolongation of APD, increased repolarization dispersion, and promotion of EAD generation. Additionally, CaMKII phosphorylates RyR2-S2814, increasing its probability of opening, which leads to abnormal Ca²⁺ release from the sarcoplasmic reticulum (SR), causing intracellular Ca²⁺ overload. This, in turn, activates NCX, generating a net inward current that induces membrane potential depolarization, forming DAD. Simultaneously, the disruption of calcium signaling significantly impacts cellular contractile function. Calcium leakage further exacerbates disturbance in calcium homeostasis by activating CaMKII, forming a vicious cycle. The dual-target combination can break the cycle and correct electrical activity abnormalities

This study further reveals that the benefits of this combined therapy stem not only from its direct synergistic effects on dual targets—electrophysiology and calcium cycling—but also likely from its ability to effectively disrupt a vicious cycle driven by oxidative stress that spans cardiomyocytes and their microenvironment. Recent studies have significantly expanded our understanding of the upstream drivers of this cycle: Zhang et al. [62] discovered that macrophages in viral myocarditis can undergo metabolic reprogramming to explosively generate mitochondrial reactive oxygen species (mtROS), while She et al. [63] elucidated that ischemia/hypoxia itself can directly cause mitochondrial damage and excessive mtROS production in cardiomyocytes. These endogenous or immune cell-derived ROS serve as potent activators of CaMKII, collectively constituting the initiating link in its excessive activation and subsequent electrophysiological disruption. The resulting calcium overload, in concert with reactive oxygen species, activates the necrotic apoptosis pathway [64], leading to cardiomyocyte death and release of injury molecules that recruit further immune cell infiltration. This forms a self-amplifying loop of “calcium overload-ROS-cell death”. Against this backdrop, the findings from this study—where combination therapy significantly improved model cell survival rates and reduced intracellular ROS levels—carry critical significance. It indicates that this therapy not only corrects electrophysiological abnormalities but also fundamentally mitigates the core driver of this vicious cycle. The GS967-dantrolene combination therapy directly targets downstream effector channels (Na_V1.5 and RyR2), achieving effective negative regulation of the hyperactive ROS/CaMKII axis by precisely correcting key pathological phenotypes downstream. This strategy aligns with the targeted antioxidant approach advocated by Ma et al. [65], while avoiding the risk of exacerbated cardiac oxidative stress from exogenous factors [66, 67]. It indirectly achieves effective negative regulation of the overactive ROS/CaMKII axis, demonstrating superior therapeutic safety.

CaMKII subtypes exhibit diversity, with the currently known subtypes mainly including α, β, γ, and δ, each of which can further form various subtype variants through alternative splicing [68]. CaMKIIδ is predominantly expressed in the heart and has multiple splice variants, such as δB, δC, and δ9. Different CaMKII-δ variants exhibit distinct or even opposing functions [69]. The δC variant promotes pathological gene expression, cell death, and arrhythmias [70], whereas the δB variant offers some protective effects [71]. Direct inhibition of CaMKII (such as with KN93), while reducing RyR2 phosphorylation [72], led to excessive shortening of FPDc, slightly below that of the CTL group (Fig. S10), suggesting a certain degree of interference with physiological electrical activity. “Global inhibitors” such as KN-93 indiscriminately inhibit all variants, suppressing the harmful δC variant while also inhibiting the potentially beneficial δB variant, which is clearly not an ideal therapeutic strategy. Additionally, the clinical translation of CaMKII antagonists has been hampered by issues such as insufficient efficacy, potential toxicity, and long-term cognitive side effects [73, 74]. In contrast, GS967-Dantrolene combination therapy directly targets downstream effector channels (Na_V1.5 and RyR2), inhibits pathological I_Na-L and calcium sparks, and indirectly modulates the CaMKII cascade (Fig. 6), thereby correcting pathological imbalances while preserving the physiological functions of CaMKII, demonstrating higher therapeutic safety. This concept is consistent with emerging evidence in cardiometabolic disease showing that transcription-factor-mediated tuning of Ca²⁺-linked stress pathways can provide targeted protection without broadly suppressing physiological signaling networks [75]. This strategy provides a novel approach for developing cardiac-specific antiarrhythmic drugs, suggesting that modulating key signaling pathways through “nodal intervention” rather than “global suppression” may be more suitable for the treatment of arrhythmias.

Although this study validated the efficacy of the dual-target strategy in hiPSC-CMs and ex vivo heart models, the following limitations should be noted. The ion channel expression profile of hiPSC-CMs (such as low expression of SCN5A and high expression of KCNH2 [76, 77]) differs from those of adult cardiomyocytes, potentially affecting drug sensitivity. However, recent evidence indicates these cells remain effective models for arrhythmia screening and drug discovery [20]. The ET-1 single-factor model cannot fully replicate the multi-mechanism-driven characteristics of clinical myocardial hypertrophy (such as mechanical stress and neuroendocrine activation). The ex vivo heart model lacks neurohumoral regulation and pharmacokinetic assessment. This study was conducted over a relatively short period, and the long-term effects of the dual-target strategy on individual cardiac function and arrhythmia risk remain unclear. In addition, MEA technology has limitations in electrophysiological assessments of hiPSC-CMs, as it cannot directly measure individual ion currents. Future research should employ techniques such as patch-clamp for direct electrophysiological recordings to precisely analyze drug effects on specific ion channels, representing a crucial research direction. Subsequent research should focus on optimizing dosing regimens, such as utilizing emerging diagnostic tools in precision medicine and nanodelivery technologies [78, 79], and exploring the therapeutic potential of this target combination in additional inherited or acquired arrhythmias, as well as validating long-term efficacy and safety in large animal models.

The study utilized PubMed as its primary database, with a search timeframe spanning from the database’s inception to 2025. The search employed a combination of core keywords centred on I_Na-L, RyR2 and arrhythmia. To our knowledge, this is the first study to propose the I_Na-L/RyR2 dual-target synergistic inhibition strategy, which offers the following advantages, reducing single-agent dosage through complementary mechanisms while minimizing off-target risks, being applicable to arrhythmias caused by various etiologies, and maintaining synergistic effects even in pathological microenvironments, aligning with complex clinical pathological features. This discovery provides a significant basis for overcoming the “efficacy-safety” bottleneck of traditional antiarrhythmic drugs and promotes a shift in the treatment paradigm from “single-target high-dose” to “multi-target low-dose” approaches.

Conclusions

This study identified a highly effective, low-toxicity dual-targeted combined inhibition therapeutic strategy targeting I_Na-L and RyR2 (GS967-Dantrolene) through screening in a hiPSC-CM arrhythmia model. In addition to direct target blockade, this combined therapy modulates signaling pathways by downregulating the CaMKII/RyR2 phosphorylation cascade. It effectively corrects pathological calcium sparks and calcium cycle dysfunction, restoring cardiomyocyte electrical signaling. Consequently, it exerts synergistic antiarrhythmic effects across multiple pathological models. These findings have significant implications for the development of novel therapeutic strategies for clinical arrhythmias.

Supplementary Information

Below is the link to the electronic supplementary material.

Abbreviations

AP

Action potential

Ang II

Angiotensin II

APD

Action potential duration

CaMKII

Ca²⁺/calmodulin-dependent protein kinase II

CaSpF

Calcium spark frequency

CICR

Calcium-induced calcium release

CSS

Combined Sensitivity Score

DAD

Delayed afterdepolarization

EAD

Early afterdepolarizations

EC₅₀

Median effect concentration

ECG

Electrocardiogram

ET-1

Endothelin-1

FPDc

Field potential duration

hiPSCs

Human-derived induced pluripotent stem cells

hiPSC-CMs

Human induced pluripotent stem cell-derived cardiomyocytes

HBSS

D-Hank’s Balanced Salt Solution

I_Ca-L

L-type calcium current

I_Kr

hERG potassium current

I_Na-L

Late sodium current

MEA

Mestro multi-well Microelectrode Array

NCX

Na⁺/Ca²⁺ exchangers

PE

Phenylephrine

PES

programmed electrical stimulation

PVDF

Polyvinylidene difluoride

ROS

Reactive oxygen species

RyR2

Ryanodine receptor 2

SCD

Sudden cardiac death

SR

Sarcoplasmic reticulum

TDP

Torsade de pointes

VF

Ventricular fibrillation

Authors’ contributions

Yanfang Xu, Qingzhong Jia and Chenxia Shi designed this study. Miaomiao Ju, Suhua Qiu, Yi Wang, Wenting Wu and Jinglei Sun conducted the study. Miaomiao Ju and Jinglei Sun analyzed the data. Miaomiao Ju and Yanfang Xu wrote the manuscript. All authors have read, commented, and agreed on the submitted version of the manuscript.

Funding

This work was supported by Central Guiding Local Science and Technology Development Fund Projects (236Z7750G).

Data availability

All the data obtained and/or analyzed in the current study were available from the corresponding authors on reasonable request.

Declarations

Ethical approval and consent to participate

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. All animal experiments were approved by the Laboratory Animal Ethical and Welfare Committee of Hebei Medical University (approval number IACUC-Hebmu-2023046).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing financial interests.