Calmodulin kinase II-mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice

Abstract

A trial fibrillation (AF), the most common human cardiac arrhythmia, is associated with abnormal intracellular Ca2+ handling. Diastolic Ca2+ release from the sarcoplasmic reticulum via "leaky" ryanodine receptors (RyR2s) is hypothesized to contribute to arrhythmogenesis in AF, but the molecular mechanisms are incompletely understood. Here, we have shown that mice with a genetic gain-of-function defect in Ryr2 (which we termed Ryr2R176Q/+ mice) did not exhibit spontaneous AF but that rapid atrial pacing unmasked an increased vulnerability to AF in these mice compared with wild-type mice. Rapid atrial pacing resulted in increased Ca2+/calmodulin-dependent protein kinase II (CaMKII) phosphorylation of RyR2, while both pharmacologic and genetic inhibition of CaMKII prevented AF inducibility in Ryr2R176Q/+ mice. This result suggests that AF requires both an arrhythmogenic substrate (e.g., RyR2 mutation) and enhanced CaMKII activity. Increased CaMKII phosphorylation of RyR2 was observed in atrial biopsies from mice with atrial enlargement and spontaneous AF, goats with lone AF, and patients with chronic AF. Genetic inhibition of CaMKII phosphorylation of RyR2 in Ryr2S2814A knockin mice reduced AF inducibility in a vagotonic AF model. Together, these findings suggest that increased RyR2-dependent Ca2+ leakage due to enhanced CaMKII activity is an important downstream effect of CaMKII in individuals susceptible to AF induction.

Figure 1. Ryr2^R176Q/+ knockin mice are vulnerable to pacing-induced AF.

(A) Representative surface ECG and intracardiac atrial electrogram showing pacing-induced AF in a Ryr2^R176Q/+ knockin mouse. Surface ECG demonstrates lack of P waves and irregular RR intervals. Atrial electrogram displays rapid and irregular atrial electrical activity. Three dots indicate truncation of the recording. (B) Continuation of the surface ECG and intracardiac atrial electrogram from A showing transition from AF to normal sinus rhythm in a Ryr2^R176Q/+ knockin mouse. P, P wave; QRS, QRS complex; A, atrial wave; V, ventricular wave. (C) Quantification of the RR interval for the beats represented in A and B showing RR variability during the irregular rhythm followed by regular RR interval during the normal sinus rhythm (NSR). Scale bar: 0.1 mV (vertical axis); 100 ms (horizontal axis).

Figure 2. CaMKII inhibition prevents pacing-induced increase in RyR2 phosphorylation at S2814.

(A) Representative Western blots of total RyR2 and S2814-phosphorylated RyR2 at baseline and after pacing in WT and Ryr2^R176Q/+ (R176Q/+) atria nontreated or treated with the CaMKII inhibitor KN-93. Bar graph showing quantification of Western blots band densities. The ratios of pS2814-RyR2 density divided by RyR2 density presented in the bar graph represent average data of 4 mice in each group. (B) Representative Western blots of total PLN and T17-phosphorylated PLN at baseline and after pacing in WT and Ryr2^R176Q/+ atria nontreated or treated with the CaMKII inhibitor KN-93. Bar graph showing quantification of Western blot band densities. The ratios of pT17-PLN density divided by PLN density presented in the bar graph represent average data of 4 mice in each group. *P < 0.05, comparing indicated groups.

Figure 3. Pharmacological and genetic inhibition of CaMKII prevents pacing-induced AF in Ryr2^R176Q/+ knockin mice.

(A) Surface ECG and intracardiac atrial electrogram showing prevention of pacing-induced AF in Ryr2^R176Q/+ mice treated with CaMKII inhibitor KN-93. Scale bar: 0.1 mV (vertical axis); 100 ms (horizontal axis). (B) Surface ECG and intracardiac atrial electrogram showing prevention of pacing-induced AF in Ryr2^R176Q/+ knockin mice expressing the CaMKII peptide inhibitor AC3-I. (C) Surface ECG and intracardiac atrial electrogram showing pacing-induced AF in Ryr2^R176Q/+ knockin mice treated with the KN-93 inactive analog KN-92. (D) Surface ECG and intracardiac atrial electrogram showing pacing-induced AF in Ryr2^R176Q/+ mice expressing the AC3-I inactive peptide analog AC3-C. (E) Bar graph summarizing percentages of mice in which AF could be induced using rapid pacing. Numbers in bars indicate numbers of mice tested in each group. *P < 0.05; **P < 0.001.

Figure 4. Pharmacological inhibition of CaMKII reverses enhanced SR Ca²⁺ leak in Ryr2^R176Q/+ atrial myocytes.

Representative [Ca²⁺]_i recordings obtained from fluo-4 AM–loaded atrial myocytes from WT (A) and Ryr2^R176Q/+ (B) mice during 1 Hz pacing in 1.8 mM Ca²⁺ (left), followed by a rapid switch to Tyrode solution containing 0 Na⁺, 0 Ca²⁺, and tetracaine (TTC) (1 mmol/l). Tetracaine was washed out 30 seconds later, and SR Ca²⁺ content was measured by changing superfusate to 10 mmol/l caffeine in 0 Na⁺, 0 Ca²⁺ Tyrode solution. SR Ca²⁺ leak was measured as shown in red and related to SR Ca²⁺ content. (C) Bar graph represents quantification of SR Ca²⁺ leak normalized to SR Ca²⁺ content in the presence and absence of KN-93 in WT and Ryr2^R176Q/+ atrial myocytes. *P < 0.05. Numbers in bars indicate numbers of myocytes tested.

Figure 5. Pacing-induced ectopic activity and reentry in Ryr2^R176Q/+ mouse atria is suppressed by pharmacological inhibition of CaMKII.

(A) Simultaneous recordings of voltage (F_v) and Ca²⁺ fluorescence (F_Ca) traces in WT mouse atria at pacing CL of 140 ms. (B) Voltage isochronal map corresponding to the paced beat 2 showing wavefront propagation across the atria (arrows) starting from the pacing electrode at the bottom (black dots). (C) Simultaneous recordings of F_v and F_Ca traces in Ryr2^R176Q/+ mouse atria at pacing CL of 140 ms demonstrating an ectopic beat occurring during the decline of the Ca²⁺ transient corresponding to beat 2, which initiates reentry. (D) Voltage isochronal map corresponding to the ectopic beat (beat 3) showing a circular propagation pattern across the atria (arrow) consistent with a reentrant circuit. The origin of the ectopic beat is indicated by an asterisk, and the circular propagation is indicated by the circular arrow. Black dots represent the position of the pacing electrodes. (E) Simultaneous recordings of F_v and F_Ca traces in Ryr2^R176Q/+ mouse atria from D after incubation with the CaMKII inhibitor KN-93. CaMKII inhibition suppresses ectopic activity and reentry at pacing CL of 140 ms. (F) Isochronal map corresponding to beat 2 shows propagation pattern across the atria from pacing electrode at the bottom (black dots). Ectopic beats and reentry were suppressed by CaMKII inhibition. Squares in B, D, and F represent the pixel at which the voltage and Ca²⁺ signals were sampled in A, C, and E, respectively.

Figure 6. Increased CaMKII-dependent phosphorylation of RyR2 at S2814 in atria of patients with chronic AF and goats with lone AF.

(A) Examples of RyR2 and phosphorylated RyR2-S2814 in atria of patients in sinus rhythm (SR) and chronic AF. Bottom: mean ± SEM protein-band intensities phosphorylated RyR2-S2814 relative to total RyR2 (n = 6 for SR and n = 6 for AF atria/analysis; *P < 0.05 versus SR). Numbers in bars indicate numbers of atria used per group. (B) Examples of total CaMKIIδ, CaMKII-pT287 (autophosphorylated), and GAPDH immunoblots. Top bands (58 kDa) represent nuclear CaMKIIδ_b and bottom (56 kDa) cytosolic CaMKIIδ_c isoforms. Bottom: mean ± SEM protein-band intensities normalized to GAPDH, expressed relative to SR (n = 7 for SR and n = 7 for AF; *P < 0.05 versus SR). (C) Examples of RyR2 and phosphorylated RyR2-S2814 in atria of goats in SR and AF. Bottom: mean ± SEM protein-band intensities phosphorylated RyR2-S2814 relative to total RyR2 (n = 4 for SR and n = 5 for AF; *P < 0.05 versus SR). (D) Examples of total CaMKII-pT287 (autophosphorylated) and calsequestrin (CSQ) immunoblots. Top bands (58 kDa) represent pT287-CaMKIIδ_b and bottom (56 kDa) pT287-CaMKIIδ_c. Bottom: mean ± SEM protein-band intensities normalized to CSQ, expressed relative to ST (n = 4 SR and n = 5 AF; *P < 0.05 versus SR).

Figure 7. Increased CaMKII-dependent phosphorylation of RyR2 at S2814 in atria of CREM-IbΔC-X transgenic mice.

Top: representative examples of RyR2 and phosphorylated RyR2-S2814 in atria of CREM-IbΔC-X transgenic and WT mice. Bottom: mean values ± SEM of protein-band intensities of phosphorylated RyR2-S2814 relative to total RyR2 (n = 8 for WT and n = 6 for CREM Tg “atria/analysis”; *P < 0.05). Numbers in bars indicate numbers of atria tested.

Figure 8. Mutation S2814A in RyR2 prevents AF in Ryr2^S2814A knockin mice.

(A) Targeted engineering of the S-to-A mutation of S2814 in the mouse RyR2 locus. A genomic clone containing exons 56 and 57 of the mouse Ryr2 gene was isolated from a 129/SvJ λKO-1 library and cloned into a pDTA4B vector using homologous recombination. (B) The S2814A mutation was introduced into exon 56 of RyR2 along with a new ClaI site. A cassette containing a loxP-flanked NeoR gene expressed from the phosphoglycerate kinase promoter (PGK-NeoR) was cloned into intron 56 to obtain the final targeting vector. (C) Targeting vector was linearized with Pme1 and electroporated into AB2.2 129Sv/J ES cells. Homologous-targeted integrands were identified using Southern blot analysis. (D) Following germline transmission and crossing with Meox2-Cre mice, final allele without Neo cassette was obtained. (E) Southern blot analysis reveals homologous-targeted mutant allele (*). (F) Heterozygous mice (HET) are bred to obtain homozygous knockin mice (HOM), identified by PCR genotyping. (G) Autoradiograph showing that CaMKII phosphorylation in the presence of γ-ATP³² was greatly inhibited in RyR2 from S2814A mouse heart. Immunoprecipitated RyR2 (input) is shown (top). Half the sample was CaMKII phosphorylated in the presence or absence of KN-93 (bottom). Noncontiguous lanes are separated by white lines. (H) Electrophysiology studies revealed that AF could be induced by cardiac pacing in 44% of WT mice after carbachol injection (50 ng/g body weight i.p.) compared with 7.7% of Ryr2^S2814A mice (*P < 0.05).