Introduction
The discovery of neuromuscular-blocking (NMB) agents marked a turning point in the history of anesthesia, leading to conceptual changes. Subsequently, separate drug groups have been adopted to achieve different anesthetic effects, which were redefined as a triad of narcosis, analgesia, and muscle relaxation [1].
Rocuronium is an aminosteroidal NMB agent that competitively antagonizes acetylcholine receptors at the neuromuscular junction [2]. It was introduced as an alternative to succinylcholine and vecuronium and has been touted as an alternative to succinylcholine for short-term surgeries and rapid sequential tracheal intubation because of its rapid onset of action [3,4]. In clinical studies, rocuronium has demonstrated reasonable cardiostability, with minimal or no effects on cardiovascular parameters related to hemodynamic effects [5]. However, rocuronium bolus administration was associated with increases in the heart rate, pulmonary vascular resistance, stroke index, and cardiac index and a decrease in pulmonary capillary wedge pressure [6]. Although rocuronium is known to increase the heart rate, its relationship to bradycardia is unclear [7]. Standard and high doses of rocuronium have been reported to cause QT interval prolongation and arrhythmias in patients with coronary artery disease [8]. Other adverse cardiac events associated with rocuronium treatment include tachycardia, cardiac arrest, circulatory collapse, ventricular fibrillation, Kounis syndrome, and stress cardiomyopathy [7].
Sugammadex is the first representative of a class of reversal drugs that functions by encapsulating steroidal NMB agents, including rocuronium, vecuronium, and pancuronium [9]. Sugammadex contains a cyclodextrin ring at the core of its molecular structure, which easily allows lipophilic molecules into its central region where they are bound by electrostatic and van der Waals interactions [10]. Therefore, sugammadex functions as a carrier for such molecules. Sugammadex has been found to elicit recovery from neuromuscular blockade more rapidly and reliably than does neostigmine, and without cholinergic side effects. However, it is also associated with adverse events, such as anaphylaxis, QT interval prolongation, severe bradycardia, grade 3 atrioventricular (AV) block, and negative pressure pulmonary edema, which can be a source of severe morbidity, in addition to common side effects, such as vomiting, dry mouth, tachycardia, dizziness, and hypotension [11–13]. Case reports of cardiac arrest after sugammadex use have also been published in recent years [14–16]. Therefore, in this study, we investigated whether sugammadex, rocuronium, and a combined sugammadex–rocuronium complex have significant effects on the electrophysiological parameters, such as action potential (AP) and contraction, of ventricular myocytes, which may be related to the cardiac and hemodynamic effects observed in clinical settings.
Materials and Methods
Study groups
After receiving approval from the Local Ethics Committee for Animal Experiments of Akdeniz University (protocol number: 940/2019.07.13), the study was performed using 3-month-old (200–250 g) male Wistar rats, with a total of 12 rats included in the experiments. The rats were kept in a controlled environment of the experimental animal unit at an appropriate temperature (22±2 °C), under a 12-h light/dark cycle with free access to food and water until used for the experiments. The rats in this study were used only for left ventricular myocyte isolation after excision of the heart. The isolated ventricular myocytes were divided into drug groups according to the NMB agents used: sugammadex, rocuronium, and the sugammadex–rocuronium combination. Drugs were applied to myocytes isolated from experimental animals, using a fast perfusion system, with a cumulative dose schedule.
Determination of experimental doses of sugammadex and rocuronium
The experimental dose of sugammadex was determined using the clinically administered dose range (2–16 mg/kg) and the corresponding plasma concentrations (0.1–197 µg/ml). Calculations using the highest clinically administered dose yielded a concentration of 9.8 × 10‒5 M, corresponding to approximately 100 µM. Accordingly, experiments were conducted using 10 µM, 100 µM, and 1 000 µM concentrations.
Similarly, the experimental dose of rocuronium was determined based on the clinically administered dose of 0.6 mg/kg, corresponding to a plasma concentration of 2 µg/ml. Calculations based on this value yielded an approximate concentration of 4 µM; therefore, experiments were conducted using 1 µM, 10 µM, and 100 µM concentrations of rocuronium.
Plasma concentrations were determined with consideration that the clinical ratio of sugammadex to rocuronium administration is 4:1. Therefore, 40 µM sugammadex and 10 µM rocuronium were considered suitable for our experimental studies.
As both sugammadex and rocuronium are in liquid form, Tyrode’s solution was used as the diluent for both compounds. Therefore, additional control experiments to assess the potential diluent effects were deemed unnecessary.
Isolation of cardiomyocytes
While the rats were under anesthesia (intraperitoneal pentobarbital sodium, 40 mg/kg), the hearts were rapidly removed and separated from excess tissue in a cold, low Ca2+ solution. To perform cell isolation using the enzymatic method, the hearts were reverse perfused via the aorta using a Langendorff perfusion system and were washed with a Ca2+-free perfusion solution containing 137 mM NaCl, 5.4 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 5.8 mM HEPES (4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid), and 20 mM glucose for 7 min. The solution was continuously bubbled with O2. Subsequently, an enzyme mixture (collagenase type 2, 0.7 mg/ml, Worthington Biochemical; and protease 0.06 mg/ml), prepared with the same dilution, was passed through the heart for 20–25 min and the heart was allowed to reach the appropriate consistency. The left ventricle was separated and cut into small pieces using scissors. The minced heart tissue was passed through a fine filter, and several washes were performed with Ca2+-free bath (extracellular) solution to remove dead cells. Finally, Ca2+ was gradually added to the cells to adapt them to physiological Ca2+ levels.
Electrophysiological recordings
Left ventricular myocytes were used in all electrophysiology experiments, and the recordings were made in a cell bath at 36 ± 1°C.
Measurement of contraction parameters
A platinum electrode was placed in the cell chamber containing the isolated ventricular myocytes, which were continuously perfused with Tyrode’s solution, for electrical field stimulation and for monitoring the contraction responses of the ventricular myocytes. The cells received 5–8 V stimuli at a frequency of 1 Hz. Changes in sarcomere length were recorded for at least 200 s until a stable response was obtained (IonOptix LLC). The contraction parameters were analyzed using IonWizard software (IonOptix LLC). Fractional shortening (L/L0 %) and contraction and relaxation velocities (µm/s) were determined and compared between drug groups.
Measurement of action potential
AP recordings were also obtained from isolated myocytes. Pipette resistances were set to 2–2.5 MΩ for all recordings. The pipette solution contained 125 mM K-aspartate, 20 mM KCl, 5 mM MgATP, 10 mM NaCl, 10 mM EGTA (Ethylene glycol-bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid), and 10 mM HEPES and was adjusted to pH 7.2 with KOH. To stimulate the cell, depolarizing pulses were applied in the current-clamp mode of the patch amplifier, and the time-dependent change in the potential was recorded to obtain AP recordings. The time to 25%, 50%, 75%, and 90% repolarization (APD25, 50, 75, 90), the resting membrane potential, and the peak AP value were measured.
Measurement of L-type Ca+2 currents
L-type Ca2+ currents (ICaL) were recorded in the whole-cell mode of the voltage-clamping technique using 2–2.5 MΩ electrodes. The pipette solution for the measurements was prepared with 120 mM L-aspartate, 20 mM CsCl, 10 mM NaCl, 5 mM MgATP, 10 mM HEPES, and 10 mM EGTA, and the pH was adjusted to 7.2 with CsOH. The standard external solution was prepared with 137 mM NaCl, 5.4 mM KCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 11.8 mM HEPES, and was adjusted to pH 7.35 with NaOH or HCl; KCl was replaced with CsCl to block K+ currents. After applying a pre-pulse of −45 mV to cells held at −70 mV to inactivate Na+ currents, 300-ms depolarizing pulses were applied in 10-mV steps from −50 mV to +60 mV to recruit the ICaL. The currents passing through a 3-kHz filter of a patch-clamp amplifier (Axon 200 B, Molecular Devices) were recorded with pClamp 10 software (Axon Instruments) at a 5-kHz sampling rate using a Digidata 1200 (Axon Instruments) and were analyzed with Clampfit 11.0.3 software (Molecular Devices). The current amplitude was calculated by subtracting the last part of the 300-ms pulse from the peak value. Finally, to eliminate the effect of cell size on the currents, each current value was divided by the capacitance of the cell, yielding the current density (pA/pF).
Measurement of potassium currents
Currents were recorded using the whole-cell configuration with the voltage-clamping method. Borosilicate glass pipettes with a resistance of 1.5–2.5 MΩ were used as electrodes for recording. A pre-pulse of −45 mV was applied to block Na+ currents, after which 3-s pulses were administered at 4-s intervals. Notably, 13 episodes were applied in 10-mV steps, from −50 mV to +70 mV, to obtain transient outward (Ito) and steady-state current values (Iss). Currents that passed through a 3-kHz filter in the voltage-clamp mode of a patch-clamp amplifier (Axon 200 B) were recorded with pClamp 10 software (Axon Instruments) at a 5-kHz sampling rate using a Digidata 1200. The extracellular solution contained 137 mM NaCl, 5.4 mM KCl, 1.5 mM CaCl2, 0.5 mM MgCl2, 10 mM glucose, and 11.8 mM HEPES (pH 7.35), while the pipette was prepared with 125 mM K-aspartate, 20 mM KCl, 5 mM MgATP, 10 mM NaCl, 10 mM HEPES, and 10 mM EGTA (pH 7.2). To block ICaL, 250 μM CdCl2 was added to the external solution. Ito was calculated by subtracting Iss at the end of the 3-s pulse from the peak value. For inward rectifier current (IK1) measurements, after the electrode was clamped to the cell; whereas, for Ito recordings, pulses were applied from −120 mV to +10 mV in steps of 10 mV for a total of 14 episodes. IK1 was calculated by measuring the current values at the end of the 3-s pulse. To eliminate the effect of cell size on the currents, current values were divided by the capacitance of the cell, yielding the current density.
The wash-out (WO) procedure
Following the bath application of rocuronium and sugammadex exposure, electrophysiological changes were allowed to stabilize for 3–5 minutes. To initiate the wash-out phase, the perfusion system was switched to a drug-free external solution (control Tyrode’s solution) maintained at room temperature and adjusted to pH 7.4. The recorded ventricular myocyte was continuously superfused with this solution at a constant flow rate of 3 ml/min using a gravity-fed system. The wash-out phase lasted for 10–15 minutes to ensure complete clearance of both compounds from the extracellular environment. Throughout this period, membrane currents and AP properties were monitored to evaluate the reversibility of drug effects. All perfusion lines and reservoirs were flushed with at least three volumes of clean solution prior to reapplication of any agent.
Statistical analysis
All statistical analyses were performed using SPSS Statistics (version 23.0; IBM Corp.). Data are presented as mean ± standard error of the mean. Normality of data distribution was assessed using the Shapiro–Wilk test. The assumption of sphericity in repeated measures was evaluated using Mauchly’s test; when this assumption was violated, the Greenhouse–Geisser correction was applied. A paired t-test was used to compare related data (baseline vs. post-intervention). Repeated-measures analysis of variance (ANOVA) was employed for comparisons involving ≥ 2 conditions across time or treatments (e.g., different concentrations of rocuronium and sugammadex, and time-dependent changes in electrical and mechanical parameters). When ANOVA detected a significant main effect, Tukey’s post-hoc multiple comparison test was applied to determine the specific group differences that contributed to the overall significance. The statistical significance level was set at 95%, where P < 0.05.
Results
Effects on contraction
Sugammadex group
Fig. 1A shows a representative example of contraction recordings, along with parameters related to fractional shortening, contraction velocity, and relaxation kinetics, obtained from 11 cardiomyocytes following the application of 10, 100, and 1000 µM sugammadex. The findings indicated that sugammadex administration causes a dose-dependent reduction in myocyte contraction amplitude (P = 0.002 for 10 µM, P < 0.001 for 100 µM and 1000 µM). In contrast, the WO procedure restored contraction amplitude to control values.
However, a significant decrease in contraction velocity was observed with the administration of sugammadex at a dose of ≥ 100 µM (P < 0.001). The effect of sugammadex on myocyte relaxation was evident for a dose of 10 µM, and this effect increased in a dose-dependent manner.
Rocuronium group
Fig. 1B shows an example of contraction recordings obtained from 8 rat ventricular myocytes after exposure to 1, 10, and 100 µM of rocuronium. No significant changes were observed in the fractional shortening rate, contraction, or relaxation kinetics according to the concentration of rocuronium (P = 0.2). These findings indicated that rocuronium did not significantly affect the mechanical function of cardiomyocytes within the tested concentration range.
Fig. 1B also summarizes the mean percentage changes of the fractional contraction recordings and contraction and relaxation parameters as compared to the control values. No statistically significant difference was observed between the values recorded for the control and the rocuronium-exposed cells (P = 0.4).
Sugammadex–rocuronium group
In the experiments, a clinically determined dose of rocuronium (10 µM) was used in combination with 40 µM sugammadex, approximately 4 times this dose, and this combination was co-administered and the effects measured in 6 ventricular myocytes. Fig. 1C shows an example of contraction recording and washing. Combination treatment suppressed myocyte contraction. In contrast, the WO reduced the peak contraction value, which returned to that recorded for the control.
Fig. 1C summarizes the percentage changes in fractional contraction recordings and the mean values of the contraction and relaxation parameters compared with the control values. A significant decrease in fractional shortening was observed (P < 0.001). Although the mean contraction values obtained after washing increased toward the control values, the recovered values remained below the control value. The contraction velocity decreased significantly (P < 0.001); however, the decrease in the relaxation rate was not statistically significant (P = 0.310). After WO, the values returned to the control values.
These findings suggest that sugammadex impairs the mechanical properties of myocytes by reducing contraction strength and slowing contraction-relaxation processes in a dose-dependent manner. In contrast, rocuronium did not appear to have a notable impact on myocyte mechanical function; however, when combined with sugammadex, it may contribute to alterations in the contraction process.
Effects of NMB agents on AP recordings
Sugammadex group
A sample recording of the AP obtained from 11 cardiomyocytes after application of 10, 100, and 1 000 µM sugammadex is shown in Fig. 2. Sugammadex had an effect on myocyte AP for a dose of ≥ 10 µM. Increasing doses demonstrated a dose-dependent effect on the AP. The calculated values for the time elapsed until the APs decreased to 25%, 50%, 75%, and 90% of the repolarization phase were termed APD25, APD50, APD75, and APD90, respectively. The mean values of these parameters are shown in Fig. 2. All doses of sugammadex significantly prolonged APD25s (P < 0.001). Among all sugammadex doses, only the 1000-µM dose caused significant prolongation of the APD25 (P = 0.020), APD50 (P < 0.001), and APD75 (P < 0.001). None of the sugammadex doses had a statistically significant effect on APD90 (P = 0.1).
Rocuronium group
A sample recording of the AP obtained from 11 ventricular myocytes after application of 1, 10, and 100 µM rocuronium is shown in Fig. 2. Rocuronium showed an effect on myocyte AP at a dose of 10 µM. Increasing doses demonstrated dose-dependent effects on the AP. The average APD25, APD50, APD75, and APD90 values of the groups are summarized in Fig. 2. A significant prolongation of all repolarization phases was seen at all rocuronium doses (APD25: P <0.001; APD50: P < 0.001; APD75: P < 0.001; APD90: P = 0.008).
Sugammadex–rocuronium group
Fig. 2 shows a representative AP recording of 7 cardiomyocytes obtained after co-administration of 40 µM sugammadex and 10 µM rocuronium. Co-administration did not change the AP durations. Fig. 2 summarizes the average APD25, APD50, APD75, and APD90 values (APD25: P = 0.4; APD50: P < 0.2; APD75: P = 0.3; and APD90: P = 0.2).
In summary, sugammadex caused a prolongation of the repolarization phase of AP, especially in the initial part (APD25) at all doses. A high sugammadex dose (1000 µM) caused a significant prolongation in all phases of repolarisation except APD90. Conversely, rocuronium caused a considerable prolongation of all AP repolarization phases at all doses (Table 1). In contrast, a combination of rocuronium encapsulated in sugammadex did not affect AP.
Effects on L-type Ca2+ currents
Sugammadex group
Fig. 3A shows a representative trace depicting the effect of sugammadex (100 µM) on ICaL recorded at 0 mV and an example of the decrease in peak current values over time after sugammadex application.
The average ICaL densities were obtained from 11 treated cells. The average values obtained as the percentage change in the peak values recorded for each cell are shown in Fig. 3A. Sugammadex (100 µM) caused a significant decrease in the peak values of calcium currents (P < 0.001).
Fig. 3B summarizes the current–voltage (I–V) characteristics of L-type calcium currents after exposure of 24 cardiomyocytes to sugammadex (100 µM). A statistically significant decrease in the current values was observed between the measured potential differences, particularly after the peak value (P < 0.001).
Rocuronium group
Fig. 4A shows an example of the effect of rocuronium (10 µM) on ICaL recorded at 0 mV and the amount of reduction in the peak current values over time. The average ICaL density is shown as a percentage change in the peak values obtained from 10 cardiomyocytes. Rocuronium (10 µM) treatment significantly decreased the peak values of calcium currents (P = 0.015).
The I–V characteristics of ICaL of 20 cardiomyocytes exposed to rocuronium (10 µM) are summarized in Fig. 4B. The decrease in the current values between the measured potential differences, particularly after the peak, was statistically significant (P < 0.001).
Sugammadex–rocuronium group
Fig. 5A presents a representative trace of the effect of a combination of 40 µM sugammadex and 10 µM rocuronium on ICaL in 11 cardiomyocytes, recorded at 0 mV. The figure also shows a reduction in the peak current values related to the current-to-cell capacitance ratio. The average ICaL densities obtained from the 11 cardiomyocytes are shown as a percentage change in the peak values in Fig. 5A. Co-administration of the drugs caused a significant decrease in peak calcium current values (P < 0.001).
The I–V characteristics of the ICaL in 22 cardiomyocytes after co-administration of the drugs are summarized in Fig. 5B. In particular, after the peak value, current values decreased significantly during the ongoing potentials (P < 0.001).
Effects on potassium currents
Sugammadex group
The I–V characteristics of the It0, Iss, and IK1 currents obtained from 24 cardiomyocytes treated with sugammadex (100 µM) are summarized in Figs. 6A and B. Significant differences between the measured potentials were observed for It0 after 40 mV and for Iss after 0 mV. No differences were found between the measured potential differences for IK1 (P = 0.2).
Rocuronium group
The I–V characteristics of the It0, Iss, and IK1 currents of 12 cardiomyocytes after rocuronium (10 µM) application are summarized in Figs. 7A and B. A significant difference between the measured potentials was observed for It0 at 40 mV (P < 0.001) (Fig. 7A). No significant difference was observed between the measured potentials of Iss and IK1 (P = 0.3).
Sugammadex–rocuronium group
Figs. 8A and B summarize the I–V characteristics of It0, Iss, and IK1 currents of 20 cardiomyocytes after the co-administration of 40 µM sugammadex and 10 µM rocuronium. The measured potentials did not differ significantly (P = 0.2).
Taken together, 100 µM sugammadex treatment caused a significant decrease in the peak values of ICaL, but had no significant effect on the inactivation–reactivation kinetics of these channels. However, this dose of sugammadex significantly suppressed the current, particularly at positive potentials. Both 10 µM rocuronium and a combination of 40 µM sugammadex and 10 µM rocuronium caused a significant decrease in the peak values of ICaL. In addition, the I–V characteristics of ICaL showed a significant decrease in current values, particularly beyond the peak value. No significant effect was observed on Ito, Iss, and IK1 currents, except for a difference with 100 µM sugammadex for It0 after 40 mV and for Iss after 0 mV, and with 10 µM rocuronium for It0 after 40 mV.
Discussion
We examined the effects of sugammadex, rocuronium, and their combination on cardiomyocyte contraction, AP duration, and ionic (calcium and potassium) currents, given that disturbances in the cardiac contractility and relaxation, Ca2+ ion influx, and AP repolarization can lead to adverse clinical events. Sugammadex can significantly modulate myocyte contractility and may reduce contractile force at higher doses. Notably, changes in contraction and relaxation velocities directly affect intracellular calcium regulation and myofibrillar contractile dynamics. A decrease in contraction velocity may be related to alterations in the cross-bridge cycling of myofilaments or a reduction in calcium influx through ion channels. Dose-dependent slowing of relaxation kinetics may be associated with changes in intracellular calcium reuptake mechanisms or a delay in calcium reuptake by the sarcoplasmic reticulum. We observed that sugammadex prolonged APD25 at all doses, whereas only a high dose prolonged APD50 and APD75. Sugammadex also suppressed calcium currents, but had no effect on potassium currents. Conversely, rocuronium had no effect on contraction, caused a significant prolongation in AP duration at all doses, and suppressed calcium flow, but had no effect on potassium flow. When both drugs were administered together, contraction was suppressed, AP duration was not prolonged, calcium currents were suppressed, but no change in potassium currents was observed.
The disturbances in the electrophysiological parameters evaluated in this study do not have specific equivalents in clinical practice. However, these are likely involved in the development of various side effects and complications. Reduced myocyte contractility diminishes the heart’s capacity to contract, resulting in inadequate blood ejection from the ventricles, which predisposes patients to decreased cardiac output and subsequent systolic heart failure [17,18]. Impaired relaxation can cause stiffening of the heart and inadequate filling during diastole, contributing to heart failure with preserved ejection fraction [19]. Disruption of the ICaL in ventricular myocytes leads to impaired cardiac electrical and mechanical functions, with serious clinical consequences. The main clinical effects of such impairment are bradycardia, malignant arrhythmias (including atrial and ventricular fibrillation and ventricular tachycardia), AV block, diastolic dysfunction, heart failure, and hypotension/cardiogenic shock [20–22]. However, the impairment of the ICaL can also trigger delayed afterdepolarization, a common precursor for arrhythmias, such as long QT syndrome [23].
Furthermore, potassium currents are vital for normal myocyte function. They are directly involved in fundamental processes, such as membrane potential maintenance, AP repolarization, excitability regulation, and muscle contraction and relaxation. Regulation of potassium currents, particularly in cardiac myocytes, is critical for maintaining the rhythm of the heart [24]. The repolarization phase is a key physiological variable that can modulate cardiac contractility and is often considered in the clinical assessment and management of arrhythmias [25]. Small changes in the ventricular AP repolarization may lead to significant physiological effects, such as the modulation of refractory periods, alterations in excitation–contraction coupling, and shifts in antiarrhythmic or proarrhythmic states, all of which play a role in various life-threatening rhythm disturbances [26,27]. Therefore, prolongation of the AP duration can lead to multiple specific clinical complications, including malignant arrhythmias, heart failure, AV block, bradycardia, and proarrhythmic states [28,29]. In our study, low doses of sugammadex affected the Ito1 and Ito2 channels, and these effects were particularly notable in the early AP repolarization phase. Sugammadex also affected ICaL, IKs, IKr, and IKI at high doses. Conversely, rocuronium affected all potassium channels responsible for AP repolarization, regardless of the dose administered. The use of rocuronium encapsulated in sugammadex did not affect AP.
Sugammadex encapsulates aminosteroidal NMB agents to form a water-soluble complex that reduces the plasma concentrations of free NMB agents. This increases the release of these agents from the nicotinic acetylcholine receptors at the motor nerve terminals, thereby restoring the muscle relaxant effect. Sugammadex does not interact directly with these receptors. Additionally, it does not affect enzyme activity and biosynthesis, and voltage-gated ion channels [30]. Sugammadex is not thought to pass into the cell, because of the large size of the gamma-cyclodextrin ring and the negative charges in its side chains, which is consistent with its low blood–brain barrier and placental transfer rate [31]. However, Kalkan et al. [32] reported that both sugammadex and rocuronium can accumulate in cardiac muscle and can cause intense edema and degeneration of myocytes.
In contrast, rocuronium competes with acetylcholine, released from motor nerve endings, for binding to postsynaptic nicotinic acetylcholine receptors. By attaching to the receptor, rocuronium blocks the ability of acetylcholine to initiate muscle cell depolarization and the associated contractile activity. Additionally, rocuronium interacts with cardiac muscarinic receptors, although with less potency than that of pancuronium, vecuronium, and pipecuronium [33]. Therefore, despite the lack of direct interaction at the receptor level, both drugs may cross the cardiac tissue, interact directly with myocytes, and exert indirect cardiac effects.
The cardiovascular safety of these drugs has been compared in many studies, using various hemodynamic parameters. Kizilay et al. [34] compared the hemodynamic parameters of sugammadex with those of neostigmine (plus atropine) in patients undergoing non-cardiac surgery and found that the sugammadex group had lower systolic, diastolic, and mean blood pressures and a slower heart rate, without any difference in QTc. They concluded that, although hemodynamic parameters increased significantly in both groups, these increases were more pronounced in patients receiving neostigmine, suggesting that sugammadex may be a safe option in this patient group. Carron et al. [35] compared the efficacy and safety of sugammadex and neostigmine in a meta-analysis of various parameters. In their study, adverse events were mainly evaluated in terms of global, respiratory, and cardiovascular effects. Sugammadex was not directly associated with a significant change in the QTc interval or other ECG abnormalities; hypotension and bradycardia were detected, but were unexplained. Hristovska et al. [36] performed a similar meta-analysis and found no significant difference between sugammadex and neostigmine in participants in terms of one or more serious or composite adverse events. However, a common feature of both meta-analyses was that neither drug was specifically evaluated for its cardiovascular side effects in the studies analyzed; rather, all possible adverse effects were considered.
The exact mechanism underlying sugammadex-induced bradycardia remains unknown. Various indirect mechanisms, particularly neural rather than humoral mechanisms of action, are thought to be involved in the cardiac effects of sugammadex, particularly bradycardia and hypotension. Due to the abrupt reversal of neuromuscular blockade and suppression of sympathetic tone, sugammadex may induce bradycardia and asystole via vagal stimulation and cholinergic dominance. The sudden return of muscle activity, particularly in the diaphragmatic and intercostal muscles, may trigger a vagal response. Rapid restoration of neuromuscular function and muscle tone may reduce compensatory sympathetic activation and alter venous tone, cardiac preload, atrial stretch, and intrinsic pacemaker activity [37]. Other possible mechanisms include the involvement of cardiac sodium and potassium channels, histamine release, autonomic shift (with a reduction in the compensatory sympathetic drive), and decreased afterload. Recently, marked bradycardia was proposed to be due to Kounis syndrome [38]. However, evidence confirming these mechanisms is lacking.
Neuromuscular blockers may induce cardiovascular effects through various mechanisms, including autonomic nervous system imbalance, histamine release, and anaphylaxis [39]. In our study, we found that rocuronium had no significant effect on myocyte contraction and potassium currents, but caused prolongation in all phases of the AP and a decrease in ICaL. Few studies have examined the effects of rocuronium on cardiac electrophysiology. Gursoy et al. [40] compared the cardiac effects of rocuronium with those of other non-depolarizing neuromuscular blockers on isolated rat atria and found that rocuronium non-significantly increased the heart rate and developed force. In a clinical study evaluating the effect of high-dose rocuronium on arrhythmia patterns in patients undergoing coronary artery bypass surgery, prolongation of QTc duration was found at doses of both 0.6 mg/kg and 1.2 mg/kg, and the authors emphasized that caution should be exercised in terms of arrhythmia development due to this rocuronium-induced prolongation [8].
Similar to sugammadex, some case reports have recounted development of Kounis syndrome due to rocuronium use [41]. Very few specific clinical and laboratory studies have addressed the direct cardiovascular effects and hemodynamic responses to rocuronium. Most studies have not examined the effects of rocuronium alone; however, its use in combination with other anesthetic agents has been evaluated. Given the limited data on the specific cardiovascular effects of rocuronium, further studies are required in this regard.
Sugammadex encapsulates rocuronium via intermolecular (van der Waals) forces, thermodynamic (hydrogen) bonds, and hydrophobic interactions, resulting in a highly robust water-soluble inclusion complex. This inclusion complex would be expected to behave similarly to sugammadex, as the rocuronium is fully encapsulated. However, allergy tests have indicated that individuals sensitive to sugammadex or rocuronium do not react to the sugammadex–rocuronium complex [42,43]. Conversely, individuals without allergic reactions to either drug can be sensitized to the complex [44]. Even when rocuronium is fully encapsulated within the cyclodextrin ring, certain parts remain accessible, which may be the source of its clinical effects [45]. This information raises the possibility that the sugammadex–rocuronium complex may produce effects distinct from those of either drug alone, potentially leading to different side effects. In our study, the sugammadex–rocuronium complex suppressed the contraction response in a manner similar to sugammadex. It decreased ICaL similarly to both drugs; however, unlike either drug alone, it did not affect K+ currents and the AP duration. Therefore, unlike sugammadex and rocuronium, the complex does not negatively affect relaxation and repolarization processes. Future in vivo and clinical studies are required to confirm these findings in physiologically and pharmacologically relevant settings.
Our study had some limitations. A single isolated myocyte may not represent the entire heart, because the ventricular myocardium comprises millions of myocytes that function as a well-connected unit or syncytium, reflecting complete interaction. Fluctuations in the AP duration can be virtually eliminated by artificially binding as few as 2 myocytes, or by deliberately selecting preparations consisting of 2 or 3 well-connected myocytes [46]. Second, the pharmacokinetic profiles of the experimental drugs should be considered when interpreting results regarding real-life effects. In clinical anesthesia practice, sugammadex is not administered alone, but is always administered after intravenous rocuronium, with variable timing, and the in vitro experimental environment cannot fully mimic the actual clinical environment in terms of its pharmacokinetic properties. Third, we administered a combined dose of sugammadex and rocuronium at a 4:1 ratio, as used in humans in clinical practice. However, sugammadex forms a 1:1 complex with rocuronium. When administered at a 4:1 ratio, the effect of the sugammadex remaining after a 1:1 combination with rocuronium may be more pronounced than the effect of the sugammadex–rocuronium complex. However, our results indicated that this theoretical possibility was unlikely, at least under our practical experimental conditions, because co-administration produced different effects than those observed with administration of the individual drugs, particularly when the effects on contraction, AP, and K+ currents were evaluated. Nevertheless, a definitive judgment could be made by observing the effects of administering the combined drugs at a 1:1 ratio. Fourth, although we deemed the use of additional diluent controls unnecessary, given the relatively high concentrations used, particularly for sugammadex (up to 1000 µM), the possibility of non-specific effects such as changes in osmolarity, pH, or ionic composition may not be completely excluded. The inclusion of a control group containing only Tyrode’s solution would help to distinguish drug-specific effects from potential dilution-related artifacts. Therefore, future studies incorporating a dedicated vehicle control group could improve the overall transparency, scientific rigor, and reproducibility of the findings.
In conclusion, our results demonstrated that sugammadex exerts a dose-dependent negative inotropic effect on cardiac myocytes and alters cardiac electrophysiological parameters, particularly by prolonging repolarization and suppressing L-type calcium currents. Rocuronium alone, while having limited mechanical effects, significantly modulated the electrophysiological properties of rat ventricular myocytes. Interestingly, the combination of sugammadex and rocuronium resulted in additive suppression of calcium currents, without significantly impacting the AP duration or potassium currents. These results suggest that the cardiac electrophysiological alterations caused by sugammadex, rocuronium, and their combination may contribute to their reported cardiovascular side effects, such as hypotension and arrhythmias. Further experimental and clinical studies are required to determine the clinical implications of these effects.
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