Calmodulin kinase II–mediated sarcoplasmic reticulum Ca2+ leak promotes atrial fibrillation in mice
Mihail G. Chelu, … , Dobromir Dobrev, Xander H.T. Wehrens
Mihail G. Chelu, … , Dobromir Dobrev, Xander H.T. Wehrens
Published June 15, 2009
Citation Information: J Clin Invest. 2009;119(7):1940-1951. https://doi.org/10.1172/JCI37059.
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Research Article Cardiology

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

Abstract

Atrial 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.

Authors

Mihail G. Chelu, Satyam Sarma, Subeena Sood, Sufen Wang, Ralph J. van Oort, Darlene G. Skapura, Na Li, Marco Santonastasi, Frank Ulrich Müller, Wilhelm Schmitz, Ulrich Schotten, Mark E. Anderson, Miguel Valderrábano, Dobromir Dobrev, Xander H.T. Wehrens

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Figure 8

Mutation S2814A in RyR2 prevents AF in Ryr2S2814A knockin mice.

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Mutation S2814A in RyR2 prevents AF in Ryr2S2814A knockin mice. (A) ...
(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 Ryr2S2814A mice (*P < 0.05).