Interleukin-6-Mediated-Ca2+ Handling Abnormalities Contributes to Atrial Fibrillation in Sterile Pericarditis Rats

Abstract

Pre-existing Ca²⁺ handling abnormalities constitute the arrhythmogenic substrate in patients developing postoperative atrial fibrillation (POAF), a common complication after cardiac surgery. Postoperative interleukin (IL)-6 levels are associated with atrial fibrosis in several animal models of POAF, contributing to atrial arrhythmias. Here, we hypothesize that IL-6-mediated-Ca²⁺ handling abnormalities contribute to atrial fibrillation (AF) in sterile pericarditis (SP) rats, an animal model of POAF. SP was induced in rats by dusting atria with sterile talcum powder. Anti-rat-IL-6 antibody (16.7 μg/kg) was administered intraperitoneally at 30 min after the recovery of anesthesia. In vivo electrophysiology, ex vivo optical mapping, western blots, and immunohistochemistry were performed to elucidate mechanisms of AF susceptibility. IL-6 neutralization ameliorated atrial inflammation and fibrosis, as well as AF susceptibility in vivo and the frequency of atrial ectopy and AF with a reentrant pattern in SP rats ex vivo. IL-6 neutralization reversed the prolongation and regional heterogeneity of Ca²⁺ transient duration, relieved alternans, reduced the incidence of discordant alternans, and prevented the reduction and regional heterogeneity of the recovery ratio of Ca²⁺ transient. In agreement, western blots showed that IL-6 neutralization reversed the reduction in the expression of ryanodine receptor 2 (RyR2) and phosphorylated phospholamban. Acute IL-6 administration to isolated rat hearts recapitulated partial Ca²⁺ handling phenotype in SP rats. In addition, intraperitoneal IL-6 administration to rats increased AF susceptibility, independent of fibrosis. Our results reveal that IL-6-mediated-Ca²⁺ handling abnormalities in SP rats, especially RyR2-dysfunction, independent of IL-6-induced-fibrosis, early contribute to the development of POAF by increasing propensity for arrhythmogenic alternans.

Keywords: Interleukin-6; alternans; calcium handling abnormalities; postoperative atrial fibrillation; ryanodine receptor.

Figure 1

IL-6 neutralization reduces AF induction and duration in SP rats. (A) Representative recordings of surface and esophageal ECG during sinus rhythm. (B) Representative recordings of AF ECG followed by burst pacing in the SP group. (C, D) Statistical results of AF duration (C) and probability (D) in the Sham, SP, IgG, and anti-IL-6 groups. n = 8/group. (E, F) Representative AP trace and ECG recorded from an SP rat, showing atrial ectopy (E) and fibrillation (F, upper panel) induced by an extrastimulus (S1S2; S2 intervals ranging from 45 to 35 ms) method. Activation maps of pacing, ectopy, reentry, and sinus rhythm (F, lower panel) corresponding to the AP trace. (G) Incidence of atrial ectopy or fibrillation for each S2 interval. Sham, n = 11; SP, n = 11; IgG, n = 8; anti-IL-6, n = 8. (H) Quantification of atrial effective refractory period. Sham n = 9; vehicle n = 8; IgG, n = 8; anti-IL-6, n = 7. Statistical analyses: χ2 test for incidence, Student t-test or one-way ANOVA with Bonferroni’s post-hoc test for the rest. ^* P < 0.05, ^** P < 0.01, ^*** P < 0.001 vs. Sham; ^### P < 0.001 vs. IgG.

Figure 2

IL-6 neutralization prevents atrial inflammation and the alterations in Ca²⁺ handling proteins in SP rats. (A, B) Representative images of atrial MPO staining (A) and quantification (B) in the 4 indicated groups. Five visual fields are taken in each sample, and the number of immune cells from these fields is quantified with ImagePro 6.0 software and is averaged to make a statistical analysis. n = 6-7/group. Scale bar: 50 µm. (C, D) Representative images of atrial CD68 staining (C) and quantification (D). n = 6-7/group. Scale bar: 50 µm. (E–I) Original Western blot (E) and quantification of the expression of RyR2, p-RyR2 (Ser2808), p-RyR2 (Ser2814) (F), the expression of SERCA and NCX (G), SERCA activity (H), the expression of PLB, p-PLB (Ser16), and p-PLB (Thr17) (I), in atrial tissue of 4 indicated groups. n =6-8/group. ^*P < 0.05, ^***P < 0.001 vs. Sham; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. IgG, determined by Student t-test or one-way ANOVA with Bonferroni’s post-hoc test. (F–H).

Figure 3

IL-6 neutralization improves atrial Ca²⁺ handling abnormalities in SP rats. (A) Atrial CaD70 maps from the 4 indicated groups at a cycle length of 142.86 ms (upper panel), and CaT traces from corresponding the 4 locations indicated with a box in the upper map (lower panel). (B) CaT traces from the 4 groups. (C) Calculation diagram of CaD. (D) Quantification of time to peak. (E, F) Quantification of CaD (E) and its coefficient of variation (F). n = 6/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Sham; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. IgG, determined by Student t-test or one-way ANOVA with Bonferroni’s post-hoc test.

Figure 4

IL-6 neutralization alleviates the onset of CaT alternans in SP rats. (A) Maps of atrial CaT alternans (spectral magnitude) in the 4 indicated groups during progressive decreases in cycle length, along with corresponding example CaT traces (amplitude normalized to large SR Ca²⁺ release to demonstrate alternan progression) from the location indicated with a box in the maps. (B) Calculation diagram of CaT alternans. (C) Quantification of CaT alternans ratio for each cycle length. n = 6/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Sham; ^#P < 0.05, ^##P < 0.01 vs. IgG, determined by Student t-test or one-way ANOVA with Bonferroni’s post-hoc test.

Figure 5

IL-6 neutralization reduces the onset of spatially discordant alternans in SP rats. (A) Maps of atrial CaT alternans (spectral magnitude) and example CaT traces from the corresponding locations in the maps at cycle lengths from 66.67 to 55.56 ms in the SP group, showing spatially concordant alternans at 66.67 ms and spatially discordant alternans (one area is out-phase with one another, accompanied by a node line in black between the two regions) at 62.5 ms, 58.82 ms, and 55.56 ms. (B) Incidence of spatially discordant alternans in the Sham, SP, IgG, and anti-IL-6 groups for each pacing rate. n = 6/group. ^*P < 0.05, ^**P < 0.01 vs. Sham; ^#P < 0.05 vs. IgG, determined by χ2 test.

Figure 6

IL-6 neutralization improves atrial RyR refractoriness in SP rats. (A) Superimposed CaT traces from the 4 indicated groups at S2 intervals ranging from 140 to 60 ms. (B) The recovery ratio of CaT (A₂/A₁) was plotted against the S2 interval at the 4 locations indicated with a box in the upper map from the 4 groups. (C) Calculation diagram of recovery ratio of CaT. (D, E) Quantification of recovery ratio of CaT (D) and its COV (E). n = 6/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Sham; ^#P < 0.05, ^##P < 0.01 vs. IgG, determined by Student t-test or one-way ANOVA with Bonferroni’s post-hoc test.

Figure 7

Exogenous IL-6 administration increases the onset of atrial CaT alternans. (A) Maps of atrial CaT alternans (spectral magnitude) in the control and IL-6 groups during progressive decreases in cycle length, along with corresponding example CaT traces from the location indicated with a box in the maps. (B) Quantification of CaT alternans for each pacing rate. (C) Superimposed CaT traces from the control and IL-6 groups at S2 intervals ranging from 140 to 60 ms. (D) Quantification of recovery ratio of CaT at each S2 interval. (E) The recovery ratio of CaT was plotted against the S2 interval at the 4 locations indicated with a box in the upper map from the 2 groups. (F) Quantification of COV of recovery ratio of CaT. Control, n = 5; IL-6, n = 6. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Control determined by Student t-test.

Figure 8

Exogenous IL-6 administration increases the alterations in Ca²⁺ handling proteins and AF induction in vivo. (A–E) Original Western blot (A) and quantification the expression of RyR2, p-RyR2 (Ser2808), p-RyR2 (Ser2814) (B), the expression of SERCA and NCX (C), SERCA activity (D), the expression of PLB, p-PLB (Ser16), and p-PLB (Thr17) (E), in atrial tissue of 2 indicated groups. n = 6/group. (F, G) Statistical results of AF duration (F) and probability (G) in the 2 indicated groups. n = 8/group. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. Control determined by Student t-test.