Enhanced sarcoplasmic reticulum Ca2+ leak and increased Na+-Ca2+ exchanger function underlie delayed afterdepolarizations in patients with chronic atrial fibrillation

Circulation. 2012 May 1;125(17):2059-70. doi: 10.1161/CIRCULATIONAHA.111.067306. Epub 2012 Mar 28.


Background: Delayed afterdepolarizations (DADs) carried by Na(+)-Ca(2+)-exchange current (I(NCX)) in response to sarcoplasmic reticulum (SR) Ca(2+) leak can promote atrial fibrillation (AF). The mechanisms leading to delayed afterdepolarizations in AF patients have not been defined.

Methods and results: Protein levels (Western blot), membrane currents and action potentials (patch clamp), and [Ca(2+)](i) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic [Ca(2+)](i) and SR Ca(2+) content (integrated I(NCX) during caffeine-induced Ca(2+) transient) were unchanged, whereas diastolic SR Ca(2+) leak, estimated by blocking ryanodine receptors (RyR2) with tetracaine, was ≈50% higher in cAF versus control. Single-channel recordings from atrial RyR2 reconstituted into lipid bilayers revealed enhanced open probability in cAF samples, providing a molecular basis for increased SR Ca(2+) leak. Calmodulin expression (60%), Ca(2+)/calmodulin-dependent protein kinase-II (CaMKII) autophosphorylation at Thr287 (87%), and RyR2 phosphorylation at Ser2808 (protein kinase A/CaMKII site, 236%) and Ser2814 (CaMKII site, 77%) were increased in cAF. The selective CaMKII blocker KN-93 decreased SR Ca(2+) leak, the frequency of spontaneous Ca(2+) release events, and RyR2 open probability in cAF, whereas protein kinase A inhibition with H-89 was ineffective. Knock-in mice with constitutively phosphorylated RyR2 at Ser2814 showed a higher incidence of Ca(2+) sparks and increased susceptibility to pacing-induced AF compared with controls. The relationship between [Ca(2+)](i) and I(NCX) density revealed I(NCX) upregulation in cAF. Spontaneous Ca(2+) release events accompanied by inward I(NCX) currents and delayed afterdepolarizations/triggered activity occurred more often and the sensitivity of resting membrane voltage to elevated [Ca(2+)](i) (diastolic [Ca(2+)](i)-voltage coupling gain) was higher in cAF compared with control.

Conclusions: Enhanced SR Ca(2+) leak through CaMKII-hyperphosphorylated RyR2, in combination with larger I(NCX) for a given SR Ca(2+) release and increased diastolic [Ca(2+)](i)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Action Potentials
  • Aged
  • Animals
  • Atrial Fibrillation / metabolism
  • Atrial Fibrillation / physiopathology*
  • Benzylamines / pharmacology
  • Biological Transport, Active
  • Caffeine / pharmacology
  • Calcium / metabolism*
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2 / metabolism
  • Calmodulin / metabolism
  • Chronic Disease
  • Female
  • Gene Knock-In Techniques
  • Humans
  • Lipid Bilayers
  • Male
  • Membrane Potentials
  • Mice
  • Myocytes, Cardiac / physiology
  • Patch-Clamp Techniques
  • Phosphorylation
  • Protein Processing, Post-Translational
  • Recombinant Fusion Proteins / metabolism
  • Ryanodine Receptor Calcium Release Channel / genetics
  • Ryanodine Receptor Calcium Release Channel / metabolism*
  • Sarcoplasmic Reticulum / metabolism*
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases / metabolism
  • Sodium-Calcium Exchanger / physiology*
  • Sulfonamides / pharmacology


  • Benzylamines
  • Calmodulin
  • Lipid Bilayers
  • Recombinant Fusion Proteins
  • Ryanodine Receptor Calcium Release Channel
  • Sodium-Calcium Exchanger
  • Sulfonamides
  • sodium-calcium exchanger 1
  • KN 93
  • Caffeine
  • Calcium-Calmodulin-Dependent Protein Kinase Type 2
  • Sarcoplasmic Reticulum Calcium-Transporting ATPases
  • Calcium