Cellular electrophysiological effects of botulinum toxin A on neonatal rat cardiomyocytes and on cardiomyocytes derived from human-induced pluripotent stem cells

Abstract

Botulinum toxin A is a well-known neurotransmitter inhibitor with a wide range of applications in modern medicine. Recently, botulinum toxin A preparations have been used in clinical trials to suppress cardiac arrhythmias, especially in the postoperative period. Its antiarrhythmic action is associated with inhibition of the nervous system of the heart, but its direct effect on heart tissue remains unclear. Accordingly, we investigate the effect of botulinum toxin A on isolated cardiac cells and on layers of cardiac cells capable of conducting excitation. Cardiomyocytes of neonatal rat pups and human cardiomyocytes obtained through cell reprogramming were used. A patch-clamp study showed that botulinum toxin A inhibited fast sodium currents and L-type calcium currents in a dose-dependent manner, with no apparent effect on potassium currents. Optical mapping showed that in the presence of botulinum toxin A, the propagation of the excitation wave in the layer of cardiac cells slows down sharply, conduction at high concentrations becomes chaotic, but reentry waves do not form. The combination of botulinum toxin A with a preparation of chitosan showed a stronger inhibitory effect by an order of magnitude. Further, the inhibitory effect of botulinum toxin A is not permanent and disappears after 12 days of cell culture in a botulinum toxin A-free medium. The main conclusion of the work is that the antiarrhythmic effect of botulinum toxin A found in clinical studies is associated not only with depression of the nervous system but also with a direct effect on heart tissue. Moreover, the combination of botulinum toxin A and chitosan reduces the effective dose of botulinum toxin A.

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Naunyn-Schmiedeberg's Archives of Pharmacology
https://doi.org/10.1007/s00210-022-02332-1
RESEARCH
Cellular electrophysiological effects ofbotulinum toxin
Aonneonatal rat cardiomyocytes andoncardiomyocytes derived
fromhuman‑induced pluripotent stem cells
AygulNizamieva1· SheidaFrolova1,2,3· MihailSlotvitsky2,3· SandaaraKovalenko2,3· ValeriyaTsvelaya2,3·
AnnaNikitina1· DavidSergeevichev4· KonstantinAgladze1,2,3
Received: 21 July 2022 / Accepted: 7 November 2022
© The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2022
Abstract
Botulinum toxin A is a well-known neurotransmitter inhibitor with a wide range of applications in modern medicine.
Recently, botulinum toxin A preparations have been used in clinical trials to suppress cardiac arrhythmias, especially in the
postoperative period. Its antiarrhythmic action is associated with inhibition of the nervous system of the heart, but its direct
effect on heart tissue remains unclear. Accordingly, we investigate the effect of botulinum toxin A on isolated cardiac cells and
on layers of cardiac cells capable of conducting excitation. Cardiomyocytes of neonatal rat pups and human cardiomyocytes
obtained through cell reprogramming were used. A patch-clamp study showed that botulinum toxin A inhibited fast sodium
currents and L-type calcium currents in a dose-dependent manner, with no apparent effect on potassium currents. Optical
mapping showed that in the presence of botulinum toxin A, the propagation of the excitation wave in the layer of cardiac cells
slows down sharply, conduction at high concentrations becomes chaotic, but reentry waves do not form. The combination
of botulinum toxin A with a preparation of chitosan showed a stronger inhibitory effect by an order of magnitude. Further,
the inhibitory effect of botulinum toxin A is not permanent and disappears after 12days of cell culture in a botulinum toxin
A-free medium. The main conclusion of the work is that the antiarrhythmic effect of botulinum toxin A found in clinical
studies is associated not only with depression of the nervous system but also with a direct effect on heart tissue. Moreover,
the combination of botulinum toxin A and chitosan reduces the effective dose of botulinum toxin A.
Keywords Botulinum toxin A· Cardiotoxicity· Antiarrhythmic effect· Cardiomyocytes
Introduction
Botulinum toxin A is a well-known neurotoxin that has
gradually found application in almost all areas of modern
medicine. Treatment with botulinum toxin A, which blocks
neuromuscular transmission and cholinergic neurotransmis-
sion, has been observed in clinical practice since the 1970s.
However, this drug is being increasingly applied to other
conditions, including cerebral palsy, cervical dystonia, col-
loid scarring, migraine, and postoperative atrial arrhythmia
(Cocco and Albanese 2018). Postoperative arrhythmia is one
of the most frequent and dangerous postoperative compli-
cations, and the antiarrhythmic use of botulinum toxin A
has been found to block the conduction of impulses along
nerve fibers without directly damaging the heart muscle.
Specifically, by blocking the release of neurotransmitters in
the synaptic cleft, botulinum toxin A disrupts the process
of neuromuscular transmission in the cardiac parasympa-
thetic ganglia. The antiarrhythmic effect of botulinum toxin
A was first shown in the heart of dogs by injection into the
atrial adipose tissue (Tsuboi etal. 2002). Further, a group of
researchers showed that the introduction of botulinum toxin
A into the heart blocked the development of atrial fibrillation
* Valeriya Tsvelaya
vts93@yandex.ru
1 Laboratory ofBiophysics ofExcitable Systems, Moscow
Institute ofPhysics andTechnology, Dolgoprudny, Russia
2 M. F. Vladimirsky Moscow Regional Research Clinical
Institute, Moscow, Russia
3 Laboratory ofExperimental andCellular Medicine, Moscow
Institute ofPhysics andTechnology, Dolgoprudny, Russia
4 “E. Meshalkin National Medical Research Center”
oftheMinistry ofHealth oftheRussian Federation, 15
Rechkunovskaya St, Novosibirsk, Russia

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caused by stimulation of the cervical vagus nerve (Oh etal.
2010). The same model showed a temporary suppression
of atrial fibrillation for 1week after the injection of botuli-
num toxin A (Oh etal. 2011). Presumably, this effect was
due to an increase in the refractory period of cardiac tissue;
however, the fundamental reasons for this phenomenon have
yet to be elucidated. Thus, botulinum toxin A can be used
in clinical practice for the treatment of postoperative atrial
fibrillation as a replacement for radiofrequency ablation of
ganglionic plexuses. Modern ablation methods are the main
type of treatment for atrial fibrillation, but they cause irre-
versible destruction of the anatomical structures of the heart
and can become proarrhythmic (Buckley etal. 2017; Lo
etal. 2013). At the same time, it has been shown that post-
operative arrhythmia is a transient phenomenon that occurs,
as a rule, in the first week after surgery (Aranki etal. 1996;
Rostagno etal. 2014). Accordingly, the temporary nature of
the effect of botulinum toxin A, shown in this work not only
on ganglia but also on cardiomyocytes, is an advantage of
the research methodology.
To date, studies on the effects of botulinum toxin A have
only been performed on neurons. The patch-clip method
showed the ability of botulinum toxin type A to inhibit
voltage-gated sodium channels of various types of isolated
neurons (Shin etal. 2011). However, research on cultured
cardiomyocytes is lacking. In 1998, a study showed that
botulinum toxin A markedly reduced the frequency of
cardiac myocytes by 2–4h in rat cardiomyocyte cultures
(Kimura etal. 1998). Studies of transmembrane currents
under the influence of other neurotoxins in isolated rat car-
diomyocytes have been carried out (Nicolas etal. 2015). A
substance that prolongs the action of botulinum toxin A,
chitosan, was discovered in a study on rats (Sergeevichev
etal. 2018). The remaining studies on the ability of botuli-
num toxin A to prevent fibrillation were purely clinical and
did not address the fundamental causes, concentrations, side
effects, or improvements in fibrillation suppression. One of
these studies was a randomized trial of patients with various
arrhythmias who were administered intravenous and epicar-
dial botulinum toxin A (Pokushalov etal. 2014, 2015). In
addition, several clinical studies have shown the promise
of using botulinum toxin A in cardiac arrhythmias (Tanyeli
and Isik 2020; Deerenberg etal. 2021). For example, it was
recently shown that when botulinum toxin A is injected into
four posterior epicardial fat pads, there is a sustained reduc-
tion in the incidence and burden of atrial tachyarrhythmias
over 3years of follow-up (Romanov etal. 2019).
This work examines the causes of the antiarrhythmic
effect of botulinum toxin type A and the substance that
prolongs its action (chitosan) and the mechanism of the
antiarrhythmic effect of botulinum toxin A on cardiomyo-
cytes. This study demonstrates the effect of the substances
on single cardiomyocytes and a monolayer of neonatal
human and rat cardiomyocytes. The fundamental mecha-
nism of suppression of the reentry wave, as a violation of
the conduction of excitation waves, is demonstrated by
assessing the likelihood of arrhythmias at different botu-
linum toxin A concentrations. Also, this paper highlights
the effect of a combination of drugs on cardiomyocytes,
namely, chitosan and botulinum toxin A. Previously,
results on the combined use of chitosan and botulinum
toxin A were shown only for animals and in clinical stud-
ies, and their effects on cells and the selection of dosages
were not considered in cell studies (Sergeevichev etal.
2020; Adler etal. 2022). In this work, we show that chi-
tosan can significantly improve the performance of botu-
linum toxin A and reduce the effective dose of the latter.
Moreover, after the combined use of botulinum toxin A
and the chitosan preparation, the cells were restored and
not damaged.
Methods andmaterials
Obtaining neonatal rat cardiomyocytes
Heart cells were isolated according to the Worthington pro-
tocol, which we used previously (Tanyeli and Isik 2020).
The hearts of Rattus Norvegicus and Sprague Dawley rats
1–4days old were used. After seeding the cells, the sam-
ples were placed in an incubator (37°C, 5% CO2) for 1–2h.
Dulbecco’s modified Eagles medium (DMEM, Gibco,
11,965,092) with 10% fetal bovine serum (FBS, Gibco,
26,140,079), referred to hereafter as DMEM-10, was then
added to each sample, and the samples were returned to the
incubator for 24h. The next day, the medium was changed
from DMEM-10 to DMEM-5.
After 3–4days of cultivation, the cells were observed
using a light microscope to detect the formation of a conflu-
ent monolayer and a contractile syncytium, which served
as an indicator of the possibility of optical mapping of this
sample. For sample preparation, pre-burned flame coverslips
(13 and 21mm in diameter for the patch-clamp and optical
mapping, respectively) were placed in 24-well culture plates
(for the patch-clamp) and in 35-mm Petri dishes (for opti-
cal mapping). The slides were then ultraviolet light (UV)
exposed for 30min. Human fibronectin (IMTEC, H Fne-C)
was used to increase the cell adhesion of neonatal rat car-
diomyocytes. Fibronectin was applied at a concentration of
20μg/ml to each coverslip, and then the petri dishes were
transferred to an incubator (37°C, 5% CO2) for 12h. Sub-
sequently, the cells were planted: for the patch-clamp drop,
strictly on the glass, they were planted at a cell concentration
of 50 thousand/cm2; for optical mapping, they were planted
at 300 thousand/cm2.

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Electrophysiological study, patch‑clamp
Whole-cell potential-dependent currents were recorded as
described previously (Frolova etal. 2016) in isolated ven-
tricular cardiomyocytes using the perforated patch-clamp
method. The study was performed on neonatal ventricular
cardiomyocytes at physiological temperatures. Amphotericin
B (Sigma, A4888) was used at a concentration of 0.24mg/
mL as a perforating agent (Lippiat 2008). Cover slides with
cultured cardiac cells were placed in a chamber mounted
on the slide of an Olympus IX71 inverted microscope. The
chamber was perfused with an extracellular solution. The
chamber solution used to record INav and ICav, L contained
10mM HEPES/NaOH (Gibco, 15,630,080), 80mM NaCl,
20mM TEA-Cl (Sigma, T2265), 10mM CsCl, 1.2mM
KH2PO4, 5mM MgSO4, 2mM CaCl2, and 20mM D-glu-
cose; pH = 7.25 (270mOsm). The pipette solution contained
10mM HEPES/NaOH, 130mM CsCl, 5mM MgSO4, and
5mM EGTA (Sigma, E4378); pH = 7.25 (285mOsm). To
record potassium currents (IK), the chamber solution con-
tained 10mM HEPES/KOH, 80mM NaCl, 5mM KCl,
1.2mM KH2PO4, 5 mM MgSO4, 2mM CaCl2, 20mM
D-glucose, pH = 7.25 (270mOsm), and a pipette solution:
10mM HEPES/KOH, 130mM KCl, 5mM MgSO4, 5mM
EGTA, pH = 7.25 (285mOsm) (Frolova etal. 2016). All
chemical components, with the exception of those indicated
separately, were supplied by RUSHIM company.
Patch-clamp pipettes were made of borosilicate glass
(Sutter Instrument, BF150-86–10) with a tip resistance
of ~ 3 MOhm when placed in the experimental solution.
The pipette displacement was corrected to zero just before
the formation of the gigaome. After the gigaomic con-
tact formation, a quick compensation adjustment of the
amplifier instrument compensated for the pipette capaci-
tance. Electrical access to the cell during perforation was
marked by the appearance of slow capacitive currents,
which increased in amplitude as pores formed in the mem-
brane, with amphotericin encapsulated in the patch pipette.
INav was recorded in response to an increasing stimulus
from − 120mV to + 50mV for 200ms, with a sustaining
potential of − 80mV (using a step at the beginning of the
stimulus: − 80 to − 120 mV for 100 ms) (Lippiat 2008).
For recording INav, a stimulus from − 80 to + 15mV, with
steps of 10mV lasting for 200ms, was also used. Current
changes in the absence and presence of the botulinum toxin
A/botulinum toxin A with chitosol were compared in the
same cardiomyocyte.
Similarly, the effect of botulinum toxin A/botulinum
toxin A with chitosol on ICav, L was examined using solu-
tions containing CsCl and TEA + to suppress Kv. A pulse
of 100ms to − 40 mV with a supporting potential (PP)
of − 80mV was used to study ICav and L currents without
interfering with INav (Sung etal. 2012). The peak of ICav, L
was measured at 0mV. A stimulus from − 60 to + 50mV, in
steps of 10mV with a duration of 500ms, was also applied.
The output IKs were induced by a 500-ms depolarizing
pulse from 0 to + 60mV (PP − 70mV). The amplitude
of the IKs was measured at the end of the voltage step
(Frolova etal. 2016).
Human iPSCs andtheir differentiation intocardiomyocytes
The following procedure was performed to obtain the
m34sk3-induced pluripotent stem cell line (iPSC) from the
monocytes of a healthy donor (Slotvitsky etal. 2020; Podgur-
skaya etal. 2019). Blood was obtained from a healthy donor
at the Novosibirsk Meshalkin Scientific Research Center,
from which a monocyte culture was isolated. The mono-
cytes were then reprogrammed to a pluripotent state using
the Epi5™ Episomal iPSC Reprogramming Kit protocol
(ThermoFisher Scientific, Invitrogen, A15960). Nucleated
fibroblasts were transferred to a culture surface coated with
Geltrex LDEV-Free hESC-Qualified Reduced Growth Fac-
tor Basement Membrane Matrix (Gibco, A1413301) in the
following culture medium: DMEM/F12 (Gibco, 11,320,033),
10% fetal bovine serum (Gibco, 26,140,079), 1 × GlutaMAX
supplement (Gibco, 35,050,061), 1 × penicillin/streptomycin
(Paneco, A063п), and 1 × non-essential amino acid solution
(Gibco, 11,140,050). The next day, the medium was changed
to DMEM/F12 medium with HEPES containing 1 × N2 sup-
plement (Gibco, 17,502,048), 1 × B27 supplement (Gibco,
A1486701), 1 × non-essential amino acids solution, 1 × Glu-
taMAX supplement, 0.1mM β-mercaptoethanol (Sigma,
M6250), and 100ng/ml bFGF (Sigma, SRP2092). Colony
formation was observed starting on day 9 following nucleo-
fection. Similar in morphology to human pluripotent cell
colonies, the resulting colonies were separated using a cap-
illary, transferred to a feeder layer (mitotically inactivated
mouse fibroblasts), and cultured in a human pluripotent
cell medium (Knockout DMEM (Gibco, 10,829,018), 15%
knockout serum replacement (Gibco, 10,828,010), 1 × Glu-
taMAX supplement, 1 × penicillin/streptomycin, 1 × non-
essential amino acids solution, 0.05mM β-mercaptoethanol,
10ng/mL basic fibroblast growth factor) to generate stable
cell lines. After several iterations of colony selection, the
cells were transferred to Geltrex and Essential 8 Medium
(ThermoFisher Scientific, Gibco, A1517001). The resulting
cell lines were fully characterized as pluripotent (Lian etal.
2013; Burridge etal. 2014).
Cell differentiation was performed according to a modi-
fied GiWi protocol based on the activation of the WNT/β-
catenin signaling pathway (by inhibiting the GSK3β pro-
tein kinase with CHIR99021) and its subsequent inhibition
(with the WNT inhibitor IWP2 (Sigma, I0536)) (Lian etal.
2013). Differentiation was started by adding a differentiation
medium (RPMI 1640, 1 × B27 supplement without insulin)

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containing a CHIR99021 concentration (Sigma, SML1046)
of 9μM to the cells for 48h. After 48h, differentiation
proceeded according to the known protocol (Burridge etal.
2014). The first cell contractions were observed on day 9 fol-
lowing differentiation. Mapping occurred when the culture
reached 50days.
Optical mapping
After 3–4days of cultivation, a monolayer of neonatal rat
cardiomyocytes was stained in the medium with 4µg/ml of
Fluo-4 (Invitrogen, F14201) fluorescent calcium-dependent
dye for 35–40min without light. All experiments were per-
formed at 37°C. After dyeing, the samples were filled with
a fresh-heated solution of Tyrode salts (Sigma, T2145) with
a pH of 7.4.
Optical mapping was performed using a setup with the
following main components: a high-sensitivity high-speed
video camera (Andor IXon3, Andor Technologies), mercury
lamp (Olympus U-RFL-T), optical microscope (Olympus
MVX10), filter cube (Olympus U-M49002XL), and uni-
versal electric pulse generator (Velleman, PCGU-1000). A
platinum point electrode and a reference circular electrode
were used for the point stimulation of the cardiac tissue.
Optical mapping of differentiated human cardiomyocytes
was performed under sterile physiological conditions in a
similar manner. Each sample was checked for the presence
of spontaneous activity. The pulse amplitude did not exceed
8V. The video was shot at 68–130 fps with a spatial resolu-
tion of ~ 0.03mm/pixel.
All solutions of the studied botulinum toxin A were
diluted in a solution of hydrogen salt. The experiments used
the proprietary Xeomin drug (Merz, 50 units per ampoule)
as botulinum toxin type A and chitosan solutions prepared
from the drug Chitosol (Koltsovo, Novosibirsk region, this
chitosan has a degree of deacetylation of at least 90% and an
average molecular weight of about 500kDa and is produced
by “Bioavanta” and prepared from crab shells). In the course
of the work, a stock solution of botulinum toxin A 1 units/μl
was used. The following dilutions were tested: 1, 0.1, 0.01,
0.001, and 0.0001 units/µl. A control was taken for each
sample. The degree of deacetylation was no less than 90%,
and the mass was 500kDa. Chitosol aqueous solutions were
obtained by dissolving succinic acid (500mg/100mL sterile
water), gradually adding chitosol (1000mg/100mL succinic
acid solution) under sonication, and sonicating the mixture
for 1h using a model UZTA-0.4/22-OM sonicator (U-sonic,
Biysk, Russia) at maximum power. Sterile water was added
to compensate for evaporation caused by prolonged sonica-
tion. The solution was filter-sterilized using 0.45-μm apy-
rogenic acetate cellulose filters (Minisart, Sartorius Stedim
Biotech Göttingen, Germany). Then, 10ml of the resulting
chitosol solution was used to dilute 100 units of botulinum
toxin A (xeomin, Merz Pharma, GmbH & Co. KGaA). Dilu-
tions of the chitosol stock solution (100μl of initial chito-
sol/10ml of Tyrode) were also tested using dilutions of 1,
0.1, 0.01, 0.001, and 0.0001 units/μL. A chitosol solution
with botulinum toxin A was made by mixing 50µl of initial
chitosol and 50µl of xeomin stock solution and then 2μl of
the resulting solution (which contains 1 unit of xeomin) was
brought up to 1ml with the chitosol stock solution.
For botulinum toxin A, the concentration range from
1 to 0.0001 units was tested. This range was necessary to
determine the effective concentrations at which the effect
on cell culture occurs in the study of cell mapping. At this
level and below, the effective influence of the concentra-
tion is understood as a significant decrease in the excit-
ability of the cell culture under the action of the desired
concentration. Based on the effective concentrations
obtained from the mapping, measurements were made on
a patch clamp. Subsequently, it was determined which of
the botulinum toxin A concentrations are effective when
used in conjunction with chitosol.
Data processing
All videos from the optical mapping were processed using
the ImageJ program. The activation and amplitude maps
were built using the Wolfram Mathematica 9 program and
Image J. Statistical significance of differences between
groups were determined using an analysis of variance
(ANOVA) followed by Fisher’s least significant difference
test for group comparison. For all results, differences of
p < 0.05 were considered significant. Data preprocessing and
normalization were performed in Microsoft Excel.
Results
Electrophysiology
To investigate the effect of 0.1 units of botulinum toxin
A on the fast sodium current of the voltage-gated chan-
nel, a stimulus in steps of 10mV lasting 200ms from − 80
to + 15mV was applied to the ventricular cardiomyocytes.
Figure1Ashows the suppression of INav by ~ 90% under
botulinum toxin A exposure. The L-type calcium current
(ICa, L) of the voltage-gated channel was activated by
applying a stimulation protocol in steps of − 40 to 0mV
for 300ms. It was shown that the L-type calcium current
(ICa, L) in the presence of 0.1 units of botulinum toxin A
was suppressed by ~ 80% (Fig.1B). Slow potassium current
IKs were obtained in response to a stimulation protocol in
steps from − 40 to + 60mV for 500ms. Exposure of the IKs
to 0.1 units of botulinum toxin A had no noticeable effect
(Fig.1C).

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In order to determine the effect of the mixture of botuli-
num toxin A and chitosol on the voltage-gated ion channels
that the botulinum toxin A suppressed (i.e., the fast sodium
current and the calcium current, L-type), the currents were
also examined in the presence of chitosol.
It was shown that chitosol alone, without botulinum toxin
A, did not affect the calcium current, L-type, ICa, L, or fast
sodium current (i.e., INav, remained uninhibited; Fig.2A)
compared to the effect of botulinum toxin A.
The voltage–current relationship showed that under the
influence of 0.01 units of botulinum toxin A with chitosol, as in
the case of 0.1 units of botulinum toxin A, INav also appeared
to be suppressed. Suppression was about ~ 95% (Fig.2Bright).
The INav stimulation protocol is shown in the figure.
We constructed current–voltage (I–V) relationships by
plotting the peak current of ICa, L elicited at each test poten-
tial normalized to cell capacitance (current density) against
the membrane potential in the control and in the presence
of 0.01 units of botulinum toxin A with chitosol (Fig.2C).
In the presence of 0.01 units of botulinum toxin A with
chitosol, calcium current L-type (ICa, L) was suppressed
by ~ 90% (Fig.2C), as in the case of botulinum toxin A alone
without chitosol, but with a higher concentration (0.1 units
botulinum toxin A).
We measured the suppression of fast sodium current
(INav) (Fig.3A top) and calcium current L-type (ICa,
L-type) (Fig.3Btop) in the presence of a lower concentra-
tion (0.001 units) of botulinum toxin A with chitosol. Sup-
pression was about 40% in the case of INav (Fig.3Abottom)
and 50% in the case of ICa, L (Fig.3Bbottom).
We also tested the effect of chitosol alone (Fig.3Cleft)
and the effect of 0.1 units of botulinum toxin A with chitosol
(Fig.3Cright) on slow potassium current IKs. There were no
significant changes in the potassium current, IKs, or amplitude.
Optical mapping ofneonatal rat cardiomyocytes
When different dilutions of botulinum toxin A were added
to the tissue culture of neonatal rat cardiomyocytes, a drop
in the velocity of excitation wave conduction was observed.
After the addition of the 0.001 units of botulinum toxin A,
there was a sharp drop in the velocity of wave propagation
in the tissue culture compared with the control (Fig.4);
then, the wave propagation velocity dropped very slightly
until the addition of 1 unit of botulinum toxin A. At the
same time, no reentry waves were formed at any of the
botulinum toxin A concentrations at any stimulation fre-
quency ranging from 1 to 5Hz.
Fig. 1 Effect of botulinum toxin A on the ionic currents of the volt-
age-gated channels of neonatal rat ventricular cardiomyocytes. A
Suppression of INav currents in the control condition (without any
substances) and in the presence of the botulinum toxin A at a con-
centration of 0.1 units. The stimulation protocol is presented in the
inset. Suppression of INav by the botulinum toxin A by approxi-
mately ~ 90%. B Overlapping recordings of ICa, L currents obtained
before and after exposure to botulinum toxin A at a concentration of
0.1 units. Suppression of ICa, L amplitude by ~ 80%. C Slow potas-
sium current IKs in the control and in the presence of botulinum toxin
A at a concentration of 0.1 units resulted in no change in the ampli-
tude of IKs

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A chitosol solution was tested separately in dilutions of
0.0001, 0.001, 0.01, 0.1, and 1 (Fig.5). However, even at
high concentrations of chitosol, the velocity of the excitation
wave in the tissue culture did not change relative to the con-
trol, remaining within 10% of the control value. The critical
frequencies when different concentrations of chitosol were
added remained the same as those for the control. In other
words, chitosol alone had no pronounced effect on the tissue.
Experiments on the addition of the chitosol solution
with botulinum toxin A showed that chitosol enhanced the
effect of botulinum toxin A (Fig.6). At a concentration
of 0.0001, there was a sharp drop in excitation velocity.
Furthermore, when subsequent dilutions were added, there
was a tendency for the excitation wave velocity to drop.
After the addition of 1 unit of chitosol solution with botu-
linum toxin A, there was a 60% drop in velocity, which
was 10% more than when 1 unit of botulinum toxin A
solution alone was added. After adding concentrations
above 0.1 units of botulinum toxin A solution with chito-
sol, no velocity drop was observed. Hence, chitosol, which
separately showed no effect on tissue culture and is not a
toxin, significantly reduced the concentration of botulinum
toxin A required for excitation suppression. No reentry
was observed when botulinum toxin A solution concentra-
tions of 0.0001 and 0.001 were added.
Optical mapping ofinduced human cardiomyocytes
According to Fig.7, the excitability of the obtained tissue
culture under the action of botulinum toxin type A decreases.
However, for some time, the excitation wave conduction rate
remains approximately the same for different concentrations.
Regarding the critical frequencies, as in the control, when
0.001 and 0.01 units of botulinum toxin A were added, the
Fig. 2 Effect of botulinum toxin
A with chitosol on the voltage-
dependent ionic currents of rat
neonatal ventricular cardiomyo-
cytes. A Effect of chitosol on
sodium ramp currents, INav,
and calcium L-type, ICa, L cur-
rents in neonatal rat ventricular
cardiomyocytes. Scaled ramp
currents recorded in response
to the same ramp protocol
(from − 120 to + 50mV, 200-ms
duration) in the control and
after chitosol addition. B (Top)
Current density–voltage (I–V)
relationship for I(Nav) obtained
in response to a depolariz-
ing step protocol (from − 80
to + 15mV, 50-ms duration) in
the control and after the addi-
tion of 0.01 units of botulinum
toxin A with chitosol. Suppres-
sion of ICa, L in the presence of
botulinum toxin A with chito-
sol ~ 80%. (Bottom) Bar chart of
the percentage of inhibition of
peak inward current amplitude
(p ≤ 0.004, n = 6). C Suppres-
sion of I(Ca,L) in the presence
of 0.01 units of botulinum toxin
A with chitosol. Normalized
current–voltage (I–V) relation-
ships of whole-cell I(Ca,L)
(top). Bar chart of the percent-
age of inhibition of peak inward
current amplitude (bottom)
(p ≤ 0.008, n = 6)

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1 3
Fig. 3 Effect of botulinum toxin
A with chitosol on the ionic cur-
rents of the voltage-gated chan-
nels of rat neonatal ventricular
cardiomyocytes. A (Top) Cur-
rent density–voltage (I–V) rela-
tionship for I(Nav) obtained in
response to a depolarizing step
protocol (from − 80 to + 15mV,
50-ms duration) in the control
and after the addition of 0.001
units of botulinum toxin A with
chitosol. (Bottom) Bar chart of
the percentage of inhibition of
peak inward current amplitude.
B Suppression of I(Ca,L) in
the presence of 0.001 units of
botulinum toxin A with chito-
sol. Normalized current–voltage
(I–V) relationships of whole-
cell I(Ca,L) (top). Bar chart of
the percentage of inhibition of
peak inward current amplitude
(bottom) (p ≤ 0.03, n = 6). C
IKs current measurement in the
presence of only chitosol (left)
and in the presence of 0.1 U of
botulinum toxin A with chitosol
(right) (p ≤ 0.03, n = 5)
Fig. 4 Decrease in the rate of excitation wave conduction in the mon-
olayer of neonatal rat cardiomyocytes as a function of botulinum
toxin A concentration. The vertical axis shows the excitation wave
conduction velocity as a percentage of the maximum control veloc-
ity. For * and **, the p-value < 0.05. The table including the data on
which the figure was compiled is presented in the additional materials
(Supplement 2). The raw data may be viewed by following the link
provided in the “Data availability” section

Naunyn-Schmiedeberg's Archives of Pharmacology
1 3
tissue culture captured all frequencies from 1 to 3.33Hz.
When a 0.1 dilution of the substance was added, the culture
stopped capturing the frequency of 3.33Hz but captured
frequencies from 1 to 2.86Hz. When a frequency of 3.33Hz
was used, the excitation wave propagated at a frequency of
1.67Hz, and no reentry occurred (which indicates the antiar-
rhythmic properties of this drug).
After the addition of botulinum toxin A, the excita-
tion wave speed gradually decreased, and the conduction
zones on the tissue cultures of human cardiomyocytes were
gradually lost. Figure8Aand B (control and with the addi-
tion of 0.01 units) show a decrease in the rate of excitation
wave conduction as the distance between the wavefronts
decreases. In Fig.8B, there is already a noticeable decrease
in the excitation wave conduction area.
The concentration of 0.1 units of botulinum toxin A can
be considered a critical threshold for adding to induced
human cardiomyocyte cultures because, after 20min of
incubation, the samples began to rapidly lose the ability to
conduct the excitation wave until a complete loss of con-
duction and no response to the external stimulus occurred.
Figure9 shows the recovery of the specimens after exposure
to botulinum toxin A. As can be seen from the figure, after
some time (12days), the effect of botulinum toxin A com-
pletely disappears, and the sample captures all the set fre-
quencies, which was not the case 24h after washing. After
24h, the monolayer did not assimilate frequencies of 2.5Hz
or higher. Again, however, reentries did not occur at any
frequency. Figure8Dand E show the recovery of sample
conductance after the complete absence of conduction and
the response to the stimulus.
Discussion ofresults
The use of botulinum toxin A as an antiarrhythmic drug
is extremely promising. There are both preclinical animal
trials (Tsuboi etal. 2002; Oh etal. 2010, 2011) and clini-
cal trials (Pokushalov etal. 2015; Romanov etal. 2019;
Sergeevichev etal. 2020; Waldron etal. 2019) show-
ing that it is a promising alternative for the treatment
of postoperative atrial fibrillation to the radiofrequency
ablation of ganglionated plexi. Moreover, the addition of
globular chitosan results in prolongation and acceleration
of the effect of botulinum toxin A in preclinical studies
(Sergeevichev etal. 2020).
Rao of conducon
velocity of the
0
0.2
0.4
0.6
0.8
1
excitaon wave, r.u.
0
B
0.001
Botulinum toxi
0.
in Aconcentrat
.01
tion, units.
0.1
Fig. 7 Decrease in the rate of excitation wave conduction in the mon-
olayer of human cardiomyocytes as a function of botulinum toxin A
concentration. All velocities are presented in relative units from the
maximum control velocity. For the control and other groups, p < 0.05.
The table including the data on which the figure was compiled is pre-
sented in the additional materials (Supplement 1). The raw data may be
viewed by following the link provided in the “Data availability” section
Relave rate of excitaon wave conducon,
0
0.2
0.4
0.6
0.8
1
1.2
fracon
0
Chitosol
c
0.1
c
oncentraon, units.
1
Fig. 5 Excitation wave conduction rate as a function of chitosol con-
centration in the monolayer of neonatal rat cardiomyocytes. The table
including the data on which the figure was compiled is presented in
the additional materials (Supplement 3). The raw data may be viewed
by following the link provided in the “Data availability” section
Fig. 6 Excitation wave velocity as a function of the concentration of chi-
tosol solution with botulinum toxin A in the monolayer of neonatal rat
cardiomyocytes. For the control and other groups (*), the p-value < 0.05.
The table including the data on which the figure was compiled is pre-
sented in the additional materials (Supplement 3). The raw data may be
viewed by following the link in the “Data availability” section

Naunyn-Schmiedeberg's Archives of Pharmacology
1 3
According to the optical mapping and electrophysiologi-
cal study results, the excitability of cardiac cell layers was
reduced under the influence of botulinum toxin A both in
rat cardiomyocyte cultures and in induced human cells. This
may be explained by the fact that the addition of botulinum
toxin A to rat cardiomyocytes results in a partial blockage
of sodium channels, as established in this study. Prior to
this patch-clamp study, botulinum toxin A studies were per-
formed exclusively on neurons, but the effect of sodium cur-
rent suppression was also present (Pokushalov etal. 2014).
In patch-clamp measurements of membrane currents in neo-
natal rat ventricular cardiomyocytes, botulinum toxin A was
shown to suppress not only fast sodium but also calcium
currents without significantly affecting potassium currents.
In this research, the decrease in calcium channel L-type cur-
rent may indicate plateau phase reduction, which leads to the
decrease in the duration of the action potential. In turn, slow
potassium channels do not change the activity at any botuli-
num toxin A concentrations or even when botulinum toxin
A combined with novochisol, although novochisol enhances
the effect of botulinum toxin A.
Experiments involving optical excitation mapping in lay-
ers of neonatal cardiomyocytes and in layers of human car-
diomyocytes obtained by cell reprogramming showed that
botulinum toxin A dose-dependently reduced the excitation
wave, up to a complete blockade. Such a complete blockade
Fig. 8 Activation maps of the excitation wave conduction in the con-
trol when the botulinum toxin A was added and after the washouts.
All maps were made in four-frame increments (2ms). Propagation of
the excitation wave: from violet to red. The red dot indicates the loca-
tion of the stimulus (8mV). A Control activation map. B Activation
map with the addition of 0.01 units of botulinum toxin A. C Activa-
tion chart after the addition of 0.1 units of botulinum toxin A. D Acti-
vation chart after 20min of washing. E Activation chart one day after
the start of washing
Fig. 9 Restoration of the excitability of a monolayer of human cardio-
myocytes before and after the cessation of the introduction of botuli-
num toxin A at different times. Shown is the change in the rate after the
addition of 0.1 units botulinum toxin A, recorded less than 1min after
addition. Then, the change in speed is presented at different time inter-
vals after washing off the substance. All speeds are presented in relative
units from the maximum reference speed. For * and **, the value of
p < 0.05. A table including the data on which the figure was compiled is
presented in the additional materials (Supplement 1). The original data
may be viewed by following the link in the “Data availability” section

Naunyn-Schmiedeberg's Archives of Pharmacology
1 3
is important, as it can be used as a form of chemical abla-
tion, which is the main mechanism of the effect of botulinum
toxin A when administered during heart surgery. In fact,
there is a new preclinical study of botulinum toxin A show-
ing this clinical effect (Piccini etal. 2022).
Further, this study demonstrates that at relatively high
concentrations of botulinum toxin A, the critical frequency
of cell layer stimulation decreased; at low concentrations,
there was no effect on the frequency. In the context of post-
operative arrhythmias associated with atrial fibrillation, this
may also indicate that the high frequencies at which fibrilla-
tion usually occurs will not be perceived by the heart tissue.
Such an effect is naturally antiarrhythmic and reduces the
incidence of reentry (Oh etal. 2011; Romanov etal. 2019;
Atienza and Jalife 2007; Honarbakhsh etal. 2019).
This study on human cardiomyocytes also showed the
effect of botulinum toxin A on the action potential duration,
as evidenced by a decrease in the critical absorbance fre-
quency of the culture when certain concentrations of botu-
linum toxin A were added. However, there was no increase
in the likelihood of reentry formation in the tissue culture.
The results confirm the action of botulinum toxin A as an
antiarrhythmic agent, as shown in clinical studies (Oh etal.
2010, 2011). It has been observed in this study that, despite
causing a decrease in conduction velocity, botulinum toxin
A does not cause an increase in the probability of reentry
(data not shown).
The most important finding is that the botulinum toxin
A is completely washed away, and the tissue culture fully
restores its functions within a few days (12days). Other sci-
entific papers claim that botulinum toxin A is washed out of
the heart within 3weeks (Oh etal. 2011). Thus, this drug
can be used to temporarily stop arrhythmia attacks with-
out subsequent cardiac conduction abnormalities. No cell
damage was observed in this study. These properties may
be useful for postoperative arrhythmias, which have been
discussed in some studies.
An interesting conclusion of this work is that there is a
substance that increases the effect of botulinum toxin A and
reduces its effective concentration by a factor of 10. This
substance is chitosol, which was presented in a clinical study
previously (Sergeevichev etal. 2018). Here, the effect and
effective doses of botulinum toxin A alone and botulinum
toxin A with chitosol were clearly demonstrated. Moreover,
it was shown that chitosol does not have its own effect on
the electrophysiology of cardiac culture.
A limitation of the study is that the experiments were
performed on ventricular cardiomyocytes. Previous research
on the action of botulinum toxin A with chitosol in the case
of ventricular fibrillation was on rat hearts (Sergeevichev
etal. 2020). However, atrial fibrillation is the most com-
mon arrhythmia following cardiac surgery, which typically
appears in the first few days after operation (Waldron etal.
2019). Studies have evaluated the antiarrhythmic properties
of botulinum toxin A using experimental models of vagus
nerve stimulation (Nazeri etal. 2017) and rapid atrial stimu-
lation (Lo etal. 2016). The authors believe that it is neces-
sary to further study the effect of botulinum toxin A with
chitosol on the electrophysiology of atrial cardiomyocytes.
The study is also limited by the fact that we used 2D
tissue hiPSC-CMs as a model of human ventricular tis-
sue. In the context of this work, this made it possible
to generalize the effect of the suppression of ionic cur-
rents on the conduction of an excitation wave in the tis-
sue. Qualitatively, this effect is consistent with clinical
results. However, the use of hiPSC-CMs monolayers
hampers the quantitative description of effects. This is
because the electrophysiology of hiPSC-CMs has fea-
tures related to the level of expression of ion channel
subunits, which affect the maximum amplitudes of ion
currents (Piccini etal. 2022) and, consequently, the rate
of excitation (Kernik etal. 2019; Kléber and Rudy 2004).
In future studies, to refine the quantitative estimates of
the decrease in conduction velocity, it would be advisable
to use cardiac tissue grown on three-dimensional fiber
matrices in order to approximate the conduction velocity
and the degree of anisotropy of the real myocardium. The
influence of a substance on the operation of ion channels
is not limited to a change in peak amplitude; in general,
it is possible to change the characteristic times channel
activation and deactivation (such effects can occur, for
example, during adreno stimulation) (O'Hara etal. 2011).
Since in this work the main focus was on the possibility
of complete (rather than partial) and reversible blocking
of fast sodium current, we did not study the effect of
botulinum toxin A on characteristic times. However, when
studying partial blocking (occurring, for example, in the
process of washing off botulinum toxin A), a dynamic
patch-clamp would be appropriate, making it possible to
analyze the amplitude and time characteristics of channels
simultaneously (Meijer van Putten etal. 2015).
In this work, the effect of botulinum toxin A was shown
on ventricular-type cardiomyocytes. This experimental
limitation is due to the fact that protocols for ventricular
differentiation are more stable and efficient than protocols
for obtaining other specific types of myocytes (atrial cells,
Purkinje fibers, pacemakers, etc.) (Sergeevichev etal.
2020). For all types of myocytes, the fast depolarization
phase is ensured by the operation of fast sodium chan-
nels (for AV node cells, the fast depolarization rate also
depends on the amplitude of the funny current), which
provides a direct relationship between the peak amplitude
of INa, the maximum depolarization rate, and, conse-
quently, the rate of excitation conduction. From this, we
can assume that the qualitative effect of botulinum toxin
A will be the same for other types of cardiomyocytes. A

Naunyn-Schmiedeberg's Archives of Pharmacology
1 3
similar role of sodium channels in different types of myo-
cytes can also be found in approaches for mathematically
modeling the transmission of excitation: detailed models of
atrial (Courtemanche etal. 1998) and ventricular (Tusscher
and Panfilov 2006) tissues use the general description of
Ina studies (Luo–Rudi formalism).
Conclusions
This study shows for the first time invitro that the combina-
tion of chitosan and botulinum toxin A increases the antiar-
rhythmic effect of botulinum toxin A on both human and rat
cardiac cells. Both substances do not cause critical damage
to either single cells or tissue cultures. The effect of botuli-
num toxin A is temporary and renewable, which is critical
for postoperative use. Further, the effective dose of botuli-
num toxin A may be lower when combined with chitosan.
Supplementary Information The online version contains supplementary
material available at https:// doi. org/ 10. 1007/ s00210- 022- 02332-1.
Acknowledgements This work was carried out within the state assign-
ment of the Ministry of Health of the Russian Federation (theme #
121031300224-1). We thank Suren Zakian’s lab for providing the hiP-
SCs of a healthy donor. This work was supported by own funds of the
Moscow Institute of Physics and Technology and M. F. Vladimirsky
Moscow Regional Research Clinical Institute, Moscow, Russia. The
work was supported by the strategic academic leadership program “Pri-
ority 2030” (Agreement 075-02-2021-1316 30.09.2021).
Author contribution All authors contributed to the study conception
and design. Sh. Frolova and S. Kovalenko performed all patch-clamp
studies. V. Tsvelaya and A. Nikitina conducted studies on human car-
diomyocytes derived from IPSCs. A. Nizamieva and M. Slotvitsky per-
formed substance studies on neonatal rat cardiomyocytes, processed all
data, and prepared graphs and figures. D. Sergeevichev and K. Agladze
wrote the main manuscript text. All authors read and approved the final
manuscript. The authors declare that all data were generated in-house
and that no paper mill was used.
Funding Ministry of Health of the Russian Federation (project
121031300224–1)
• D. Sergeevichev
M. F. Vladimirsky Moscow Regional Research Clinical Institute,
Moscow, Russia (own fundings)
• Sh. Frolova
• S. Kovalenko
• M. Slotvitsky
• K. Agladze
Moscow Institute of Physics and Technology (own fundings, strategic
academic leadership program “Priority 2030”)
• V. Tsvelaya
• A. Nizamieva
• A. Nikitina
The funders had no role in study design, data collection and inter-
pretation, or the decision to submit work for publication.
Data availability The raw/processed data required to reproduce these
findings could be found at: https:// drive. google. com/ drive/ folde rs/
1EmvS YCro6 3dlE2 0vXIj HQ- tHpsU JpF65
Declarations
Ethical approval This study was performed in line with the principles
of the Declaration of Helsinki and the Guide for the Care and Use of
Laboratory Animals, published by the United States National Institutes
of Health (Publication No. 85–23, revised 1996), and was approved by
the Moscow Institute of Physics and Technology Life Science Center
Provisional Animal Care and Research Procedures Committee, Proto-
col #A2-2012–09-02.
The cell line m34Sk3 is provided by the “E. Meshalkin National Medi-
cal Research Center” of the Ministry of Health of the Russian Fed-
eration and handling approved by the Institute of Circulation Pathol-
ogy Ethics Committee (#27, March 21, 2013). All experiments and
procedures were performed in accordance with principles for human
experimentation as defined in the 1964 Declaration of Helsinki and its
later amendments and were approved by the Scientific Council of the
MIPT Life Science Center.
Consent to participate Informed consent was obtained from all indi-
vidual participants included in the study.
Consent for publication The authors affirm that human research partic-
ipants provided informed consent for publication of all figures, tables,
and data included in this article.
Competing interests The authors declare no competing interests.
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