Chronic Kidney Disease Increases Atrial Fibrillation Inducibility: Involvement of Inflammation, Atrial Fibrosis, and Connexins

Figures

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  Figure 1. Inducible AF by transesophageal atrial pacing. (A) Schematic diagram of AF induction. AF was induced by a 30-s burst pacing protocol at a 20-ms cycle length, 5-ms pulse width, and 50-h frequency. AF was considered as positive if an episode of rapid, quivering atrial beating with an irregular heart rhythm lasting over 5-s, was induced. (1) Before burst pacing, P-waves are clearly distinguishable with a regular heart rhythm; (2) Burst stimuli were delivered; (3) After burst pacing, P-waves were disappeared and replaced by rapid, quivering, and sawtooth-like waves (the f-waves) with an irregular heart rhythm, suggesting an episode of AF; (4) f-waves are dispelled by P-waves, representing the termination of AF and the recovery of sinus rhythm. (B) AF inducibility and duration. Data show that CKD results in significant higher AF inducibility and longer AF duration compared with the sham group. AF was induced three times for acquiring the standard deviation of AF inducibility. AF inducibility is shown as mean + SD and the duration is shown as median [2.5–97.5 percentile]. n = 10 and 15 in Sham and CKD group, respectively. AF, atrial fibrillation; SR, sinus rhythm.

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  Figure 2. Electrogram morphology. P-wave duration and PR interval were measured from an electrogram (ECG, lead II) before burst pacing. (A,B). Representative ECG recordings from the sham and the CKD group. In the sham group, P-waves are unidirectional whereas P-waves are biphasic and prolonged in CKD group. (C) Data suggested that P-wave duration and the ratio of P-wave duration/PR interval (P/PR) are significantly increased in the CKD group than those in the sham group while PR intervals revealed no difference. Data are shown as mean + SD. n = 8 and 9 in Sham and CKD group, respectively.

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  Figure 3. Atrial interstitial fibrosis by Sirius red-fast green counter stain. (A) Microscopy ( × 200) shows that atrial interstitial fibrosis was dyed red and myocytes dyed green. Atrial myocytes seem to be more diffused and hypertrophic in CKD rats compared with the sham group. (B) A marked increase in atrial interstitial fibrosis was found in CKD rats compared with the sham group. Data are shown as mean + SD. n = 5 and 6 in Sham and CKD group, respectively. Scale bar = 50 μm. LAA, left atrial appendage; LAS, left atrial septum.

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  Figure 4. TGFβ1/Smad2/3 signal pathway is activated in CKD rats. (A) Expression of TGFβ1, p-Smad2, and p-Smad2, n = 4 per group; (B) Expression of collagen type I (Col I), α-SMA from fresh left atria tissues, n = 4 per group; (C) Immunohistochemistry of Col I and α-SMA, n = 6 per group, magnifications × 200, scale bar = 100 μm. LAA, left atrial appendage; LAS, left atrial septum.

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  Figure 5. NLRP3 inflammasome/caspase-1/IL-1β and IL18 axis is activated in CKD rats. (A) Protein expressions of NLRP3, ASC, and pro-caspase-1 (pro-csp1); (B) Protein expressions of the matured caspase-1, the P20 and P10; (C) Protein expressions of the matured IL-1β and IL-18. Tissue was collected from the left atrium. n = 4 per group. ASC, apoptosis-associated speck-like protein containing a carboxy-terminal CARD; Csp-1 P10, active caspase-1 (10 kDa subunit); Csp-1 P20, active caspase-1 (20 kDa subunit); IL-1β, interleukin-1β; IL-18, interleukin-18; NLRP3, NLR (nucleotide-binding domain leucine-rich repeat-containing receptor) pyrin domain-containing protein 3; pro-csp1, pro-caspase-1.

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  Figure 6. Increased Rac-1, CTGF and N-Cadherin expression in LA of rats with CKD. (A) Protein amount of Rac-1, n = 4 per group; (B,C). Immunohistochemistry of CTGF and N-cadherin, n = 6 per group, magnifications × 200, scale bar = 100 μm. CTGF, connective tissue growth factor; Rac1, Ras-related C3 botulinum toxin substrate 1; LAA, left atrial appendage; LAS, left atrial septum.

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  Figure 7. Protein amount of Cx40 and Cx43 by western blot. Shown are protein expressions of Cx40, non-phosphorylated-Cx43 (np-Cx43) and phosphorylated-Cx43 (p-Cx43). These results show that CKD resulted in a decrease in Cx40 and p-Cx43 and an increase in np-Cx43. n = 4 per group.

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  Figure 8. Immunohistochemistry staining of Cx40 and Cx43. The protein level of Cx40 was lower but Cx43 higher in the CKD group compared with the sham group in both LAA and LAS (data are shown in the results section). Additionally, the distribution of both proteins are more lateralized in the CKD group than those in the sham group. Magnifications × 400, scale bar = 20 μm. LAA, left atrial appendage; LAS, left atrial septum. (→) indicates lateralized Cx40 or Cx43.