Distinct Effects of Ibrutinib and Acalabrutinib on Mouse Atrial and Sinoatrial Node Electrophysiology and Arrhythmogenesis
What Is New? The Bruton tyrosine kinase inhibitor, ibrutinib, impairs sinoatrial node and atrial conduction by blocking ionic currents in the sinoatrial node (rapid delayed rectifier K + current) and atria (Na + current and transient outward K + current), while a related, second‐generation inhibitor, acalabrutinib, does not affect these currents. What Are the Clinical Implications? These findings provide an explanation for the occurrence of atrial fibrillation and sinoatrial node dysfunction in patients receiving ibrutinib, but not acalabrutinib, for the treatment of B‐cell lymphomas. 

The Bruton tyrosine kinase inhibitor, ibrutinib, impairs sinoatrial node and atrial conduction by blocking ionic currents in the sinoatrial node (rapid delayed rectifier K + current) and atria (Na + current and transient outward K + current), while a related, second‐generation inhibitor, acalabrutinib, does not affect these currents. 

These findings provide an explanation for the occurrence of atrial fibrillation and sinoatrial node dysfunction in patients receiving ibrutinib, but not acalabrutinib, for the treatment of B‐cell lymphomas. 

The data from this study and the materials used are available from the corresponding author upon reasonable request. Mice This study was conducted using male C57BL/6 mice between the ages of 8 and 15 weeks. All experimental procedures were approved by the Animal Care and Use Committees of the University of Calgary and Western University and were in accordance with the guidelines of the Canadian Council on Animal Care. In Vivo Electrophysiology and Arrhythmia Studies Mice received an acute dose of either acalabrutinib (10 mg/kg) or vehicle (trappsol) by oral gavage followed by an intracardiac electrophysiology study 90 minutes after drug delivery. This dose was selected to directly compare with previous studies demonstrating that similar, clinically relevant doses of ibrutinib lead to pacing‐induced AF. This dose of acalabrutinib would yield higher serum concentrations compared with ibrutinib, ensuring that any effects on AF would be identified. Acalabrutinib was solubilized in trappsol dissolved in distilled water and delivered by oral gavage in a single dose. Intracardiac studies were performed as we have previously described. , Arrhythmia induction used both programmed electrical stimulation and burst pacing (2‐ms pulses at 50 Hz, 400‐ms burst duration) to determine susceptibility to AF (rapid atrial activity lasting >1 second) as well as atrial effective refractory period, ventricular effective refractory period, atrioventricular node effective refractory period, and Wenckebach cycle length. Additional information is available in Data . High‐Resolution Optical Mapping Activation patterns and electrical conduction in the atria and right atrial (RA) posterior wall (ie, SAN region) were investigated using high‐resolution optical mapping in isolated atrial preparations as previously described. , , Atrial preparations were immobilized using blebbistatin (10 µmol/L). Changes in fluorescence were captured using the voltage‐sensitive dye RH‐237 (15 µmol/L) and a CMOS camera (SciMedia) at 1000 frames per second. Experiments were conducted in sinus rhythm and during pacing at 8 Hz. All experiments were performed at 37 °C. Data were analyzed using custom software written in MATLAB (MathWorks). Further details are available in Data . Patch‐Clamping of Isolated Atrial and SAN Myocytes RA and SAN myocytes were isolated from mice by enzymatic digestion as we have previously described. , , These myocytes were used to record APs and ionic currents by whole‐cell patch‐clamp at room temperature (21–23 °C). The solutions and experimental protocols for each of these approaches are available in Data . Drug Doses In optical mapping and patch‐clamp studies, ibrutinib was primarily delivered acutely at concentrations of 0.1 µmol/L or 10 µmol/L. The lower dose (0.1 µmol/L) was used in some experiments to determine whether lower concentrations of ibrutinib produced similar effects to the higher dose. Note that in some experiments in isolated SAN myocytes, ibrutinib was used at an even lower dose of 0.05 µmol/L, which was necessary to observe effects without complete suppression of AP firing. Acalabrutinib was used at concentrations of 10 µmol/L and 50 µmol/L in isolated tissues. The dose of 10 µmol/L was used to directly compare with ibrutinib while the higher dose was used to ensure that an absence of effects at 10 µmol/L was not dose related. These concentrations of ibrutinib and acalabrutinib span a range that is expected to approximate those achieved at the doses used for in vivo studies. Statistical Analysis All data are expressed as mean±SEM. Statistical analysis was conducted using Prism version 8.3.1 (Graphpad Software). Data were analyzed using Fisher exact test, paired Student t test, 1‐way repeated measures ANOVA with a Tukey post hoc test, 2‐way repeated measures ANOVA with a Tukey post hoc test, or a mixed effects analysis with a Tukey post hoc test as indicated in the figure legends. Mixed effects analysis was used in instances when a value was missing from a data set, which prevented the use of 1‐way or 2‐way ANOVAs. P <0.05 was considered statistically significant. 

This study was conducted using male C57BL/6 mice between the ages of 8 and 15 weeks. All experimental procedures were approved by the Animal Care and Use Committees of the University of Calgary and Western University and were in accordance with the guidelines of the Canadian Council on Animal Care. 

Mice received an acute dose of either acalabrutinib (10 mg/kg) or vehicle (trappsol) by oral gavage followed by an intracardiac electrophysiology study 90 minutes after drug delivery. This dose was selected to directly compare with previous studies demonstrating that similar, clinically relevant doses of ibrutinib lead to pacing‐induced AF. This dose of acalabrutinib would yield higher serum concentrations compared with ibrutinib, ensuring that any effects on AF would be identified. Acalabrutinib was solubilized in trappsol dissolved in distilled water and delivered by oral gavage in a single dose. Intracardiac studies were performed as we have previously described. , Arrhythmia induction used both programmed electrical stimulation and burst pacing (2‐ms pulses at 50 Hz, 400‐ms burst duration) to determine susceptibility to AF (rapid atrial activity lasting >1 second) as well as atrial effective refractory period, ventricular effective refractory period, atrioventricular node effective refractory period, and Wenckebach cycle length. Additional information is available in Data . 

Activation patterns and electrical conduction in the atria and right atrial (RA) posterior wall (ie, SAN region) were investigated using high‐resolution optical mapping in isolated atrial preparations as previously described. , , Atrial preparations were immobilized using blebbistatin (10 µmol/L). Changes in fluorescence were captured using the voltage‐sensitive dye RH‐237 (15 µmol/L) and a CMOS camera (SciMedia) at 1000 frames per second. Experiments were conducted in sinus rhythm and during pacing at 8 Hz. All experiments were performed at 37 °C. Data were analyzed using custom software written in MATLAB (MathWorks). Further details are available in Data . 

RA and SAN myocytes were isolated from mice by enzymatic digestion as we have previously described. , , These myocytes were used to record APs and ionic currents by whole‐cell patch‐clamp at room temperature (21–23 °C). The solutions and experimental protocols for each of these approaches are available in Data . 

In optical mapping and patch‐clamp studies, ibrutinib was primarily delivered acutely at concentrations of 0.1 µmol/L or 10 µmol/L. The lower dose (0.1 µmol/L) was used in some experiments to determine whether lower concentrations of ibrutinib produced similar effects to the higher dose. Note that in some experiments in isolated SAN myocytes, ibrutinib was used at an even lower dose of 0.05 µmol/L, which was necessary to observe effects without complete suppression of AP firing. Acalabrutinib was used at concentrations of 10 µmol/L and 50 µmol/L in isolated tissues. The dose of 10 µmol/L was used to directly compare with ibrutinib while the higher dose was used to ensure that an absence of effects at 10 µmol/L was not dose related. These concentrations of ibrutinib and acalabrutinib span a range that is expected to approximate those achieved at the doses used for in vivo studies. 

All data are expressed as mean±SEM. Statistical analysis was conducted using Prism version 8.3.1 (Graphpad Software). Data were analyzed using Fisher exact test, paired Student t test, 1‐way repeated measures ANOVA with a Tukey post hoc test, 2‐way repeated measures ANOVA with a Tukey post hoc test, or a mixed effects analysis with a Tukey post hoc test as indicated in the figure legends. Mixed effects analysis was used in instances when a value was missing from a data set, which prevented the use of 1‐way or 2‐way ANOVAs. P <0.05 was considered statistically significant. 

Effects of Acalabrutinib on Atrial Electrophysiology In Vivo Previously, our group showed that acute administration of ibrutinib (10 mg/kg) significantly increases AF susceptibility in mice. To assess whether acalabrutinib had similar effects on AF susceptibility, intracardiac electrophysiology studies were conducted in mice given a single acute dose of acalabrutinib (10 mg/kg) or vehicle control. No AF was observed in any mice (12 given acalabrutinib and 14 given vehicle) ( and Figure ). Furthermore, acalabrutinib had no significant effects on other measures of atrial or cardiac electrophysiology including atrial effective refractory period, Wenckebach cycle length, atrioventricular nodal effective refractory period, or ventricular effective refractory period (Table ). These data indicate that, unlike ibrutinib, acalabrutinib is not associated with increased AF susceptibility. Effects of Ibrutinib and Acalabrutinib on Atrial Electrophysiology in Isolated Atrial Preparations Next, we sought to directly assess the effects of ibrutinib and acalabrutinib on atrial electrical conduction patterns and atrial optical AP (OAP) morphology using high‐resolution optical mapping in isolated atrial preparations. Representative activation maps (Figure ) and OAPs from the right and left atria (Figure ) demonstrate that conduction time across the atria was slowed and OAPs were prolonged following superfusion with ibrutinib. Beating rate in atrial preparations was dose‐dependently reduced in the presence of ibrutinib (0.1 µmol/L and 10 µmol/L). This effect was partially reversible upon washout but remained reduced compared with baseline (Figure ). Ibrutinib also dose‐dependently decreased right (Figure ) and left (Figure ) atrial CV in atrial preparations in sinus rhythm. In addition, ibrutinib dose‐dependently prolonged RA (Figure ) and left atrial (Figure ) APD, measured at 70% repolarization in atrial preparations in sinus rhythm. The effects of ibrutinib on atrial CV and APD were reversible upon drug washout. The effects of ibrutinib on atrial electrophysiology were also measured in atrial preparations paced at 8 Hz to account for rate‐dependent effects. The effects of ibrutinib on atrial CV and APD in paced preparations were similar to those observed in sinus rhythm (Figure ). Next, the effects of acalabrutinib on atrial electrophysiology were investigated at doses of 10 µmol/L and 50 µmol/L. In contrast to ibrutinib, representative activation maps (Figure ) demonstrate similar conduction patterns and conduction times before and after application of acalabrutinib. Representative OAPs (Figure ) show that acalabrutinib resulted in APD prolongation. There were no effects of acalabrutinib on atrial preparation beating rate (Figure ). Furthermore, acalabrutinib had no significant effects on right (Figure ) or left (Figure ) atrial CV in atrial preparations in sinus rhythm at either dose. Nevertheless, APD at 70% repolarization was prolonged in both atria following superfusion of acalabrutinib, particularly at the higher dose (Figure ). The effects of acalabrutinib on atrial CV and APD were similar in atrial preparations paced at 8 Hz (Figure ). Effects of Ibrutinib and Acalabrutinib on Atrial Myocyte Electrophysiology To further investigate the basis for the distinct effects of ibrutinib and acalabrutinib on atrial electrophysiology, AP morphology (Figure ) was measured in isolated RA myocytes by patch‐clamping. Ibrutinib (10 μmol/L) had no significant effect on resting membrane potential (Figure ) but significantly reduced V max and overshoot (Figure ; Table ). Consistent with optical mapping studies, ibrutinib also prolonged APD throughout repolarization in isolated atrial myocytes (Figure , Table ). These effects were reversible upon washout. In contrast, acalabrutinib (10 µmol/L and 50 µmol/L doses) had no significant effects on resting membrane potential, V max , or overshoot (Figure , Table ). The lower dose of acalabrutinib (10 µmol/L) also had no significant effect on APD; however, it did prolong APD at the higher dose (50 µmol/L) (Figure , Table ). To determine the basis for the changes in atrial AP morphology, the effects of ibrutinib and acalabrutinib on ionic currents were investigated. Because ibrutinib reduced AP V max in atrial myocytes, we measured I Na following acute application of ibrutinib (10 µmol/L; Figure ). I Na I‐V curves demonstrate that ibrutinib reduced I Na density (Figure ) in association with a reduction in I Na conductance as measured from steady‐state activation curves (Figure ). These effects of ibrutinib on I Na density and conductance were reversed upon washout. While conductance was reduced, ibrutinib had no significant effects on I Na voltage for 50% channel activation (V 1/2(act) ) (Figure ) or slope factor (Figure ). Ibrutinib also had no significant effects on I Na steady‐state inactivation (Figure ). In contrast, acalabrutinib had no significant effects on I Na current density or I Na conductance even at the higher 50 µmol/L dose (Figure ). To investigate the basis for APD prolongation, we next measured repolarizing K + currents between −120 mV and +80 mV using voltage‐clamp protocols with and without a prepulse to inactivate I to , (Figure ). Summary I‐V curves illustrate that ibrutinib (10 µmol/L) reduced peak outward total K + current (I K(tot) ) when recorded with and without a prepulse (Figure ). Furthermore, the difference in current between these 2 recordings demonstrates that ibrutinib reduced I to (Figure ). These effects were reversible upon washout. The effects of ibrutinib on I Kur , measured as the component of I K(tot) sensitive to 4‐aminopyridine (4‐AP), were also assessed by superfusing atrial myocytes with ibrutinib followed by 4‐AP (100 µmol/L) in the presence of ibrutinib (Figure ). Ibrutinib reduced outward I K(tot) , and subsequent application of 4‐AP had no further effect indicating that ibrutinib inhibited the 4‐AP–sensitive I Kur (Figure ). Ibrutinib had no significant effects on inward rectifier K + current as seen at membrane potentials more negative than −80 mV (Figure ). Because only 50 μmol/L of acalabrutinib prolonged APD, we also investigated the effects of this dose of acalabrutinib on I k(tot) (Figure ). Summary I‐V curves demonstrate that acalabrutinib reduced peak outward I K(tot) when recorded with and without an inactivating prepulse (Figures ); however, there was no effect of acalabrutinib on I to (Figure ). Similar to ibrutinib, acalabrutinib reduced the 4‐AP–sensitive I Kur in atrial myocytes (Figure ). Acalabrutinib had no significant effects on inward rectifier K + current (Figure ). As APD can also be affected by I Ca,L , the effects of ibrutinib (10 µmol/L) on I Ca,L in RA myocytes were measured (Figure ). These data demonstrate that ibrutinib had no significant effects on atrial I Ca,L density, I Ca,L conductance, or I Ca,L steady‐state activation properties (Figure ). Effects of Ibrutinib and Acalabrutinib on SAN Electrophysiology in Isolated Atrial Preparations Given that beating rate in isolated atrial preparations was reduced by ibrutinib, but not acalabrutinib, the effects of both drugs on SAN electrophysiology were further investigated using optical mapping (Figure ). Activation maps, as well as OAPs from the SAN in the region of the leading activation site and in the adjacent atrial myocardium (first atrial activation adjacent to the CT in the RA free wall) were measured (Figure ). These were used to analyze CV in the SAN region of the RA posterior wall as well as SAN to atrial conduction time. Ibrutinib dose‐dependently reduced CV in the RA posterior wall and increased SAN to atrial conduction time (Figure ). Ibrutinib also increased SAN APD at 50% repolarization (Figure ). These effects of ibrutinib were reversible upon washout. In contrast, acalabrutinib had no significant effects on CV, SAN to atrial conduction time, or APD at 50% repolarization in the SAN when applied at doses of 10 µmol/L (to directly compare with the higher dose of ibrutinib) or an even higher dose of 50 µmol/L (Figure ). Effect of Ibrutinib on Isolated SAN Myocyte Electrophysiology The ability of ibrutinib to decrease beating rate and modulate SAN conduction in isolated atrial preparations indicates direct effects on the SAN. This was further investigated by measuring the effects of ibrutinib on spontaneous APs in isolated SAN myocytes. Strikingly, 10 μmol/L of ibrutinib fully arrested regular spontaneous AP firing in SAN cells in association with a depolarization of the MDP (Figure ). Only low‐amplitude oscillating membrane potentials between −20 mV and −30 mV, which could not be recovered following up to 45 minutes of washout, were seen (Figure ). To observe more modest effects on SAN myocytes that did not result in complete suppression of AP firing, it was necessary to use a lower concentration of ibrutinib of 0.05 µmol/L. At this lower dose, ibrutinib reduced SAN myocyte spontaneous AP firing (Figure ) in association with a depolarization of the MDP (Figure ), prolongation of APD at 50% repolarization (Figure ), and a reduction in DD slope (Figure ). Other measures of SAN AP morphology are provided in Table . Conversely, acalabrutinib (10 μmol/L) had no significant effects on SAN AP firing or morphology (Figure , Table ). To determine the basis for the effects of ibrutinib on SAN AP firing frequency, MDP, and APD, we measured repolarizing K + currents under voltage clamp conditions in isolated SAN myocytes before and after application of ibrutinib (10 μmol/L). Similar to atrial myocytes, summary I‐V curves illustrate that ibrutinib reduced outward I K(tot) in isolated SAN myocytes (Figure ). In contrast, there was no effect of acalabrutinib (10 µmol/L) on outward I k(tot) in SAN myocytes (Figure ). I Kr plays an essential role in regulating APD, MDP, and AP firing rate in the SAN, including in mice. Thus, the effects of ibrutinib (10 µmol/L) on I Kr were measured in SAN myocytes using voltage‐clamp protocols designed to detect I Kr tail currents (Figure ). Ibrutinib again reduced peak outward I K during the test pulses (measured between −50 mV and +40 mV; Figure ). Furthermore, Boltzmann fitting of I Kr tail currents (measured at −45 mV) illustrate that ibrutinib reduced I Kr density (Figure ) in association with a negative shift in the V 1/2(act) (Figure ). The lower dose of ibrutinib (0.05 µmol/L) had similar effects on I Kr in SAN myocytes (Figure ). In contrast, and consistent with the lack of an effect on APD and I k(tot) , acalabrutinib (10 µmol/L) had no significant effects on I Kr in SAN myocytes (Figure , ). Ibrutinib (10 µmol/L) had no significant effects on hyperpolarization activated current amplitude or activation kinetics in isolated SAN myocytes (Figure ). Ibrutinib also had no significant effects on I Ca,L density or activation kinetics in SAN myocytes (Figure ). 

Previously, our group showed that acute administration of ibrutinib (10 mg/kg) significantly increases AF susceptibility in mice. To assess whether acalabrutinib had similar effects on AF susceptibility, intracardiac electrophysiology studies were conducted in mice given a single acute dose of acalabrutinib (10 mg/kg) or vehicle control. No AF was observed in any mice (12 given acalabrutinib and 14 given vehicle) ( and Figure ). Furthermore, acalabrutinib had no significant effects on other measures of atrial or cardiac electrophysiology including atrial effective refractory period, Wenckebach cycle length, atrioventricular nodal effective refractory period, or ventricular effective refractory period (Table ). These data indicate that, unlike ibrutinib, acalabrutinib is not associated with increased AF susceptibility. 

Next, we sought to directly assess the effects of ibrutinib and acalabrutinib on atrial electrical conduction patterns and atrial optical AP (OAP) morphology using high‐resolution optical mapping in isolated atrial preparations. Representative activation maps (Figure ) and OAPs from the right and left atria (Figure ) demonstrate that conduction time across the atria was slowed and OAPs were prolonged following superfusion with ibrutinib. Beating rate in atrial preparations was dose‐dependently reduced in the presence of ibrutinib (0.1 µmol/L and 10 µmol/L). This effect was partially reversible upon washout but remained reduced compared with baseline (Figure ). Ibrutinib also dose‐dependently decreased right (Figure ) and left (Figure ) atrial CV in atrial preparations in sinus rhythm. In addition, ibrutinib dose‐dependently prolonged RA (Figure ) and left atrial (Figure ) APD, measured at 70% repolarization in atrial preparations in sinus rhythm. The effects of ibrutinib on atrial CV and APD were reversible upon drug washout. The effects of ibrutinib on atrial electrophysiology were also measured in atrial preparations paced at 8 Hz to account for rate‐dependent effects. The effects of ibrutinib on atrial CV and APD in paced preparations were similar to those observed in sinus rhythm (Figure ). Next, the effects of acalabrutinib on atrial electrophysiology were investigated at doses of 10 µmol/L and 50 µmol/L. In contrast to ibrutinib, representative activation maps (Figure ) demonstrate similar conduction patterns and conduction times before and after application of acalabrutinib. Representative OAPs (Figure ) show that acalabrutinib resulted in APD prolongation. There were no effects of acalabrutinib on atrial preparation beating rate (Figure ). Furthermore, acalabrutinib had no significant effects on right (Figure ) or left (Figure ) atrial CV in atrial preparations in sinus rhythm at either dose. Nevertheless, APD at 70% repolarization was prolonged in both atria following superfusion of acalabrutinib, particularly at the higher dose (Figure ). The effects of acalabrutinib on atrial CV and APD were similar in atrial preparations paced at 8 Hz (Figure ). 

To further investigate the basis for the distinct effects of ibrutinib and acalabrutinib on atrial electrophysiology, AP morphology (Figure ) was measured in isolated RA myocytes by patch‐clamping. Ibrutinib (10 μmol/L) had no significant effect on resting membrane potential (Figure ) but significantly reduced V max and overshoot (Figure ; Table ). Consistent with optical mapping studies, ibrutinib also prolonged APD throughout repolarization in isolated atrial myocytes (Figure , Table ). These effects were reversible upon washout. In contrast, acalabrutinib (10 µmol/L and 50 µmol/L doses) had no significant effects on resting membrane potential, V max , or overshoot (Figure , Table ). The lower dose of acalabrutinib (10 µmol/L) also had no significant effect on APD; however, it did prolong APD at the higher dose (50 µmol/L) (Figure , Table ). To determine the basis for the changes in atrial AP morphology, the effects of ibrutinib and acalabrutinib on ionic currents were investigated. Because ibrutinib reduced AP V max in atrial myocytes, we measured I Na following acute application of ibrutinib (10 µmol/L; Figure ). I Na I‐V curves demonstrate that ibrutinib reduced I Na density (Figure ) in association with a reduction in I Na conductance as measured from steady‐state activation curves (Figure ). These effects of ibrutinib on I Na density and conductance were reversed upon washout. While conductance was reduced, ibrutinib had no significant effects on I Na voltage for 50% channel activation (V 1/2(act) ) (Figure ) or slope factor (Figure ). Ibrutinib also had no significant effects on I Na steady‐state inactivation (Figure ). In contrast, acalabrutinib had no significant effects on I Na current density or I Na conductance even at the higher 50 µmol/L dose (Figure ). To investigate the basis for APD prolongation, we next measured repolarizing K + currents between −120 mV and +80 mV using voltage‐clamp protocols with and without a prepulse to inactivate I to , (Figure ). Summary I‐V curves illustrate that ibrutinib (10 µmol/L) reduced peak outward total K + current (I K(tot) ) when recorded with and without a prepulse (Figure ). Furthermore, the difference in current between these 2 recordings demonstrates that ibrutinib reduced I to (Figure ). These effects were reversible upon washout. The effects of ibrutinib on I Kur , measured as the component of I K(tot) sensitive to 4‐aminopyridine (4‐AP), were also assessed by superfusing atrial myocytes with ibrutinib followed by 4‐AP (100 µmol/L) in the presence of ibrutinib (Figure ). Ibrutinib reduced outward I K(tot) , and subsequent application of 4‐AP had no further effect indicating that ibrutinib inhibited the 4‐AP–sensitive I Kur (Figure ). Ibrutinib had no significant effects on inward rectifier K + current as seen at membrane potentials more negative than −80 mV (Figure ). Because only 50 μmol/L of acalabrutinib prolonged APD, we also investigated the effects of this dose of acalabrutinib on I k(tot) (Figure ). Summary I‐V curves demonstrate that acalabrutinib reduced peak outward I K(tot) when recorded with and without an inactivating prepulse (Figures ); however, there was no effect of acalabrutinib on I to (Figure ). Similar to ibrutinib, acalabrutinib reduced the 4‐AP–sensitive I Kur in atrial myocytes (Figure ). Acalabrutinib had no significant effects on inward rectifier K + current (Figure ). As APD can also be affected by I Ca,L , the effects of ibrutinib (10 µmol/L) on I Ca,L in RA myocytes were measured (Figure ). These data demonstrate that ibrutinib had no significant effects on atrial I Ca,L density, I Ca,L conductance, or I Ca,L steady‐state activation properties (Figure ). 

Given that beating rate in isolated atrial preparations was reduced by ibrutinib, but not acalabrutinib, the effects of both drugs on SAN electrophysiology were further investigated using optical mapping (Figure ). Activation maps, as well as OAPs from the SAN in the region of the leading activation site and in the adjacent atrial myocardium (first atrial activation adjacent to the CT in the RA free wall) were measured (Figure ). These were used to analyze CV in the SAN region of the RA posterior wall as well as SAN to atrial conduction time. Ibrutinib dose‐dependently reduced CV in the RA posterior wall and increased SAN to atrial conduction time (Figure ). Ibrutinib also increased SAN APD at 50% repolarization (Figure ). These effects of ibrutinib were reversible upon washout. In contrast, acalabrutinib had no significant effects on CV, SAN to atrial conduction time, or APD at 50% repolarization in the SAN when applied at doses of 10 µmol/L (to directly compare with the higher dose of ibrutinib) or an even higher dose of 50 µmol/L (Figure ). 

The ability of ibrutinib to decrease beating rate and modulate SAN conduction in isolated atrial preparations indicates direct effects on the SAN. This was further investigated by measuring the effects of ibrutinib on spontaneous APs in isolated SAN myocytes. Strikingly, 10 μmol/L of ibrutinib fully arrested regular spontaneous AP firing in SAN cells in association with a depolarization of the MDP (Figure ). Only low‐amplitude oscillating membrane potentials between −20 mV and −30 mV, which could not be recovered following up to 45 minutes of washout, were seen (Figure ). To observe more modest effects on SAN myocytes that did not result in complete suppression of AP firing, it was necessary to use a lower concentration of ibrutinib of 0.05 µmol/L. At this lower dose, ibrutinib reduced SAN myocyte spontaneous AP firing (Figure ) in association with a depolarization of the MDP (Figure ), prolongation of APD at 50% repolarization (Figure ), and a reduction in DD slope (Figure ). Other measures of SAN AP morphology are provided in Table . Conversely, acalabrutinib (10 μmol/L) had no significant effects on SAN AP firing or morphology (Figure , Table ). To determine the basis for the effects of ibrutinib on SAN AP firing frequency, MDP, and APD, we measured repolarizing K + currents under voltage clamp conditions in isolated SAN myocytes before and after application of ibrutinib (10 μmol/L). Similar to atrial myocytes, summary I‐V curves illustrate that ibrutinib reduced outward I K(tot) in isolated SAN myocytes (Figure ). In contrast, there was no effect of acalabrutinib (10 µmol/L) on outward I k(tot) in SAN myocytes (Figure ). I Kr plays an essential role in regulating APD, MDP, and AP firing rate in the SAN, including in mice. Thus, the effects of ibrutinib (10 µmol/L) on I Kr were measured in SAN myocytes using voltage‐clamp protocols designed to detect I Kr tail currents (Figure ). Ibrutinib again reduced peak outward I K during the test pulses (measured between −50 mV and +40 mV; Figure ). Furthermore, Boltzmann fitting of I Kr tail currents (measured at −45 mV) illustrate that ibrutinib reduced I Kr density (Figure ) in association with a negative shift in the V 1/2(act) (Figure ). The lower dose of ibrutinib (0.05 µmol/L) had similar effects on I Kr in SAN myocytes (Figure ). In contrast, and consistent with the lack of an effect on APD and I k(tot) , acalabrutinib (10 µmol/L) had no significant effects on I Kr in SAN myocytes (Figure , ). Ibrutinib (10 µmol/L) had no significant effects on hyperpolarization activated current amplitude or activation kinetics in isolated SAN myocytes (Figure ). Ibrutinib also had no significant effects on I Ca,L density or activation kinetics in SAN myocytes (Figure ). 

This study provides new insight into the basis for ibrutinib‐mediated AF, as well as sinus bradycardia and sinus arrest, following ibrutinib treatment. We demonstrate the effects of ibrutinib on atrial and SAN electrophysiology that are consistent with increased AF susceptibility and SAN dysfunction and provide insight into the ionic basis for these effects. Furthermore, this study demonstrates that several of these effects are specific to ibrutinib and that another more selective BTK inhibitor, acalabrutinib, does not elicit the same effects. Thus, we have shown that ibrutinib and acalabrutinib have distinct effects on atrial electrophysiology and arrhythmogenesis that explain why ibrutinib is proarrhythmic while acalabrutinib is not. We previously demonstrated that acute application of ibrutinib in vivo resulted in increased susceptibility to pacing‐induced AF in mice. Consistent with this, we now show that ibrutinib dose‐dependently reduced atrial CV as assessed using high‐resolution optical mapping. These effects were elicited by acute drug application and could largely be reversed upon washout. Consistent with these impairments in conduction, ibrutinib reduced AP V max and atrial I Na without altering I Na activation kinetics. In stark contrast, acalabrutinib had no effects on atrial AP V max or I Na . Atrial I Na and AP V max are critical determinants of atrial CV. Furthermore, slow conduction increases susceptibility to AF by shortening the wavelength for reentry. , Based on this, our data indicate that ibrutinib increases AF occurrence by impairing atrial conduction via its effects on I Na . Consistent with the absence of effects on I Na , we show that acalabrutinib did not increase susceptibility to AF in mice in vivo, further supporting the conclusion that the presence or absence of effects on I Na and atrial conduction are key determinants of AF susceptibility in the presence of ibrutinib compared with acalabrutinib. We also found that ibrutinib and acalabrutinib each increased atrial APD; however, these effects occurred by distinct mechanisms. Ibrutinib prolonged APD by blocking I to and I Kur , while acalabrutinib increased APD by blocking I Kur only. Furthermore, it took higher doses of acalabrutinib to increase APD and the effects were still smaller in magnitude compared with ibrutinib, which could be caused by the effects of ibrutinib on both I to and I Kur . Increases in APD could increase the likelihood of early afterdepolarizations and conduction block, each of which can favor AF initiation or maintenance. , Thus, more substantial AP prolongation, along with reductions in AP V max , could contribute to AF occurrence in the presence of ibrutinib, while more modest increases in APD alone are insufficient to increase AF susceptibility in the presence of acalabrutinib. Previous studies have shown that chronic ibrutinib treatment in mice is associated with enhanced Ca 2+ /calmodulin‐dependent protein kinase II (CaMKII) activity and changes in C‐terminal Src kinase (CSK) activity. , These studies further show that chronic ibrutinib led to atrial enlargement, atrial fibrosis, inflammation, and changes in sarcoplasmic reticulum Ca 2+ handling as a result of these alterations in CaMKII and CSK activity. While BTK is expressed in the heart, , these findings, in conjunction with our study, support the hypothesis that ibrutinib increases AF susceptibility via off‐target effects independent of BTK inhibition. This is further supported by our finding that acalabrutinib, which is a more selective BTK inhibitor, has minimal effects on atrial electrophysiology and no effects arrhythmogenesis. Whether CaMKII and/or CSK mediate any of the effects of ibrutinib on atrial myocyte electrophysiology is unknown, and ibrutinib may also modulate other signaling pathways in atrial myocytes. Our observations that ibrutinib, but not acalabrutinib, can inhibit multiple currents (I Na and I to ), and that I Na is reduced without changes in activation kinetics, may indicate that these effects involve direct binding of ibrutinib to these ion channels. On the other hand, ibrutinib and acalabrutinib both inhibited I Kur similarly, suggesting that this effect could involve BTK inhibition or effects on another common signaling pathway. These hypotheses could be investigated in future studies. The implications of atrial enlargement and fibrosis in AF occurrence during ibrutinib treatment are not completely clear given we have previously shown that AF susceptibility is reduced after a 24‐hour washout of ibrutinib. This suggests that the rapidly activatable effects of ibrutinib on atrial ion channels that we have identified are centrally involved in ibrutinib‐mediated atrial arrhythmogenesis. Nevertheless, atrial enlargement and fibrosis can both contribute importantly to conduction disturbances and reentry in AF ; therefore, these effects of ibrutinib could further enhance susceptibility to arrythmia along with changes in atrial ion channel function. Interestingly, 1 study suggested that long‐term treatment with ibrutinib did not affect atrial AP morphology; however, this study delivered ibrutinib for 4 weeks in vivo and then conducted patch‐clamp studies in isolated myocytes, which were compared with a vehicle control group. Based on our data, it is possible that the effects of ibrutinib on atrial myocyte electrophysiology could have been washed out during the cell isolation procedure, causing the effects of ibrutinib on cellular atrial electrophysiology to go undetected. Our study indicates that it is critical to consider the acute effects of ibrutinib on cellular electrophysiology and ion channel function when interpreting the effects of ibrutinib on AF occurrence. In addition to promoting AF, ibrutinib has also been associated with sinus bradycardia and even sinus arrest , ; however, the basis for this was completely unknown. We found that ibrutinib consistently decreased beating rate in association with impaired conduction in the RA posterior wall and increases in SAN to atrial conduction time in isolated atrial preparations, while acalabrutinib had no effects on these properties. Using isolated SAN myocytes, we also show that ibrutinib, but not acalabrutinib, slowed spontaneous AP firing in SAN myocytes in association with depolarization of the MDP, a reduction in DD slope, and increases in APD. At higher doses, ibrutinib completely suppressed AP firing in association with substantial depolarization of the MDP. Interestingly, these effects of ibrutinib did not involve hyperpolarization activated current or I Ca,L . Rather, ibrutinib potently inhibited I Kr , which has previously been shown to play an important role in SAN AP firing. , Specifically, inhibition of I Kr in SAN myocytes with compounds such as dofetilide or E‐4031 leads to effects similar to those observed with ibrutinib, including depolarization of the MDP, reduction in DD slope, and increases in APD, all of which can contribute to a reduction in spontaneous AP firing. , , , These findings indicate that ibrutinib has selective effects on I Kr in SAN myocytes that account for the effects on AP morphology and reductions in heart rate. These effects are also likely to be independent of BTK as acalabrutinib had no effect on heart rate or I Kr in SAN myocytes. Some limitations should be noted for our study. Our experiments were performed in mice, which exhibit some differences in ion channel expression and function compared with humans. For example, I Kr contributes to atrial repolarization in large mammals and humans, but not in mice. Interestingly, I Kr is an important ionic current in mouse SAN and we have demonstrated that it is potently affected by ibrutinib in SAN myocytes. As such, future studies on the role of I Kr in ibrutinib‐mediated AF are warranted. In addition, our study focused on acute effects of ibrutinib and acalabrutinib. While no AF was observed during acute acalabrutinib application, the effects of chronic acalabrutinib treatment on atrial electrophysiology were not studied. In addition to being affected by I Na , atrial conduction is also affected by gap junction conductance, which was not investigated in the present study. Patch‐clamp studies were not performed in left atrial myocytes; however, optical mapping studies demonstrate that the effects of ibrutinib and acalabrutinib in right and left atria were similar. Accordingly, it is expected that drug effects on ion channels demonstrated in isolated RA myocytes would be similar in left atrial myocytes. In conclusion, our study provides novel insight into the electrophysiological effects of the BTK inhibitors ibrutinib and acalabrutinib on atrial electrophysiology and AF susceptibility. We demonstrate that ibrutinib and acalabrutinib have distinct effects on atrial conduction, atrial AP morphology, and atrial ion channel function. We also demonstrate distinct effects of these BTK inhibitors on SAN beating rate and SAN ion channel function. These data provide an explanation for the occurrence of AF and SAN dysfunction in patients treated with ibrutinib compared with those treated with acalabrutinib. 

This work was supported by the Canadian Institutes of Health Research to R.A.R. (MOP 142486 and PJT 1666105) and a Canadian Cardiovascular Society Bayer Resident Vascular Research Award to J.M.T. L.J.B. holds a University of Calgary Silver Anniversary fellowship. T.W.D. holds a Canadian Institutes of Health Research Doctoral Research Award. H.J.J. was supported by a Killam Postdoctoral Fellowship and holds a Libin Cardiovascular Institute Postdoctoral Fellowship. Y.L. holds a Canadian Institutes of Health Research Postdoctoral Fellowship. 

None. 

Data S1 Tables S1–S4 Figures S1–S7 Click here for additional data file. 

