Phosphorylated Connexin 43 is Associated with Increased Susceptibility for Atrial Fibrillation in Mice Treated with a Single High Dose of Ibrutinib but not Acalabrutinib

Abstract

Ibrutinib, an irreversible inhibitor of Bruton’s tyrosine kinase is a highly effective treatment of B‐Cell lymphoproliferative disorders. However, therapy has been shown to increase the incidence of Atrial Fibrillation (AF) in patients. Second generation drugs such as Acalabrutinib have been suggested to have less AF propensity, although there may be a signal for enhanced ventricular arrhythmias. Previously, we demonstrated an increased susceptibility to electrically induced AF with acute Ibrutinib treatment in mice. As conduction slowing due to alterations in connexin (Cx) content or phosphorylation state have been associated with AF, we hypothesised that such Cx changes would be one mechanism underlying AF susceptibility. Experiments followed the Canadian Council on Animal Care Guidelines for the Care and Use of Laboratory Animals, and the protocol was approved by the Animal Care Committee of Western University. Using previously published techniques, western blot was used to determine if a single dose by oral gavage (10 mg/kg), of Ibrutinib or Acalabrutinib reduced the content of Cx43 and/or Cx40 and/or the phosphorylated forms of Cx43 (S368 and Y265), increase of which have been shown to decrease Cx43 permeability and pore closure. Two‐month old C57BL/6, male (n=18), and female mice (n=18), were given Trappsol vehicle control, Ibrutinib or Acalabrutinib by oral gavage. After 90 min, they were anaesthetized with an intraperitoneal (IP) mixture of Ketamine (100 mg/kg) and Xylazine (10 mg/kg). The heart was removed under anaesthesia, blotted to remove excess blood, the atria separated and weighed before rapid freezing in liquid nitrogen for later homogenization and protein analysis. Proteins were separated on polyacrylamide SDS gels, transferred to Polyvinylidene fluoride (PVDF) membranes and reacted with specific Cx antibodies followed by specific secondary antibodies linked to horseradish peroxidase (HRP). Content was detected by enhanced chemiluminescence (ECL). The content was expressed as the ratio to α‐actinin, as an internal loading control. Statistical evaluation was done with 2‐way ANOVA followed by post‐hoc Tukey test. Single dose Ibrutinib or Acalabrutinib did not alter the protein content of Cx43 or the content of Cx43 (Y265) or its ratio to total Cx43 in either male or female mice. However, the content of Cx43 (S368) and the ratio of the phosphorylated to total Cx43 content was higher in Ibrutinib but not the Acalabrutinib treated mice. Further the content of Cx40 was higher in Ibrutinib treated female than male mice. There was a treatment by sex interaction. These preliminary results suggests that off‐target phosphorylation at the S368 site of Cx43 may be one contributor to the acute pro‐arrhythmic effect of Ibrutinib, the magnitude of which may be sex‐dependent. The process by which this occurs and the lack of Acalabrutinib effect as well as potential ventricular targets that may contribute to ventricular arrhythmia requires further investigation.