Abstract
Aim: We investigated the underlying mechanisms in atrial fibrillation (AF) associated with R33Q mutation and Ca2+-triggered activity.Methods and Results: We examined AF susceptibility with intraesophageal burst pacing in the sarcoplasmic reticulum (SR) Ca2+ leak model calsequestrin 2 R33Q (Casq2R33Q/R33Q) mice. Atrial trigger appeared in R33Q mice but not WT mice (17.24%, 5/29 vs. 0.00%, 0/32, P < 0.05). AF was induced by 25 Hz pacing in R33Q mice (48.27%, 14/29 vs. 6.25%, 2/32, P < 0.01). The mice were given 1.5 mg/kg isoproterenol (Iso), and the incidences of AF increased (65.51%, 19/29 vs. 9.21%, 3/32, P < 0.01). Electrophysiology experiments and the recording of intracellular Ca2+ indicated significant increases in the Ca2+ sparks (5.24 ± 0.75 100 μM-1.s-1 vs. 0.29 ± 0.04 100 μM-1.s-1, n = 20, P < 0.05), intracellular free Ca2+ (0.238 ± 0.009 μM vs. 0.172 ± 0.006 μM, n = 20, P < 0.05), Ca2+ wave (11.74% vs. 2.24%, n = 20, P < 0.05), transient inward current (ITi) (-0.56 ± 0.02 pA/pF vs. -0.42 ± 0.01 pA/pF, n = 10, P < 0.05), and oscillation in membrane potentials (10.71%, 3/28 vs. 4.16%, 1/24, P < 0.05) in the R33Q group, but there was no significant difference in the L-type calcium current. These effects were enhanced by Iso, and the inhibition of calmodulin-dependent protein kinase II (CaMKII) by 1 μM KN93 reversed the effects of Iso on Ca2+ sparks (5.01 ± 0.66 100 μm-1.s-1 vs. 11.33 ± 1.63 100 μm-1.s-1, P < 0.05), intracellular Ca2+ (0.245 ± 0.005 μM vs. 0.324 ± 0.008 μM, P < 0.05), Ca2+ wave (12.35% vs. 17.83%, P < 0.05), ITi (-0.61 ± 0.02 pA/pF vs. -0.78 ± 0.03 pA/pF, n = 10, P < 0.05), and oscillation in membrane potential (17.85% 5/28 vs. 32.17% 9/28, P < 0.05). The reduction of ryanodine receptor 2 (RyR2) stable subunits (Casq2, triadin, and junctin) rather than RYR2 and the increase in CaMKII, phosphor-CaMKII, phosphor-RyR2 (Ser 2814), SERCA, and NCX1.1 was reflected in the R33Q group.Conclusion: This study demonstrates that the increase in spontaneous calcium elevations corresponding to ITi that may trigger the oscillation in membrane potentials in the R33Q group, thereby increasing the risk of AF. The occurrence of spontaneous calcium elevations in R33Q atrial myocytes is due to the dysfunction of RyR2 stable subunits, CaMKII hyperactivity, and CaMKII-mediated RyR phosphorylation. An effective therapeutic strategy to intervene in Ca2+-induced AF associated with the R33Q mutation may be through CaMKII inhibition.
Highlights
Atrial fibrillation (AF) is one of the most common arrhythmias
It is well known that Ca2+ is mainly released from the sarcoplasmic reticulum (SR) by the ryanodine receptor 2 (RyR2) channel, but the number of RyR2 channels in patients with chronic AF is significantly reduced or unchanged (Tang et al, 2007; Greiser and Schotten, 2013), and it is difficult to explain why spontaneous calcium elevations (SCaE) increase in AF
Our study showed that the baseline intracellular Ca2+ level was higher in the R33Q
Summary
Atrial fibrillation (AF) is one of the most common arrhythmias. In a canine atrial tachyarrhythmia model, it was reported that intracellular Ca2+ overload occurs during AF, and that the intracellular Ca2+ concentration subsequently returns to normal after AF (Akar et al, 2003). The increased sensitivity of Ca2+ release channels indicates the preformation of spontaneous calcium elevations (SCaE), consisting of Ca2+ sparks and Ca2+ waves associated with a high level of intracellular Ca2+ content (Wier et al, 1987). It was reported that “hyperphosphorylation” of RyR2 appears to be the main factor responsible for unstable channels (Voigt et al, 2012). RYR2 phosphorylation was unaltered in a patient with paroxysmal AF, whereas the RYR2 single-channel open probability was increased (Voigt et al, 2014). The RyR2 stable subunits include calsequestrin 2 (Casq2), triadin, and junctin, all of which are involved in the regulation of Ca2+ release (FranziniArmstrong et al, 2005). Casq2-R33Q mice display an adaptive pattern, and the sensitivity of RyR2s from Casq2R33Q mice was between that of Casq2−/− and WT mice (Valle et al, 2014). Few studies have been performed that examine the association of Casq2-R33Q and AF, and whether there is the same action on AF with Casq2−/− and Casq2-R33Q mutations is uncertain, given the lack of support of experiments
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