| Background and Aim: Atrial fibrillation(AF)is the common rapid atrial arrhythmia.Intracellular Ca2+ disorders was found in atrial myocytes of chronic AF patients,but it is unknown whether Spontaneous Ca2+ release event(SCa E)is a cause of chronic AF.In this study,we used Ca2+ disorder model-Calsequestrin 2 mutation mice(Calsequestrin 2 R33Q/R33 Q or CASQ2R33Q/R33 Q or R33Q)to investigate Ca2+ triggered activity and its underlying mechanisms in atrial fibrillation(AF).Methods: We examined AF susceptibility with intra-esophageal burst pacing in sarcoplasmic reticulum(SR)Ca2+ leak model CASQ2 R33 Q / R33Q(R33Q)mice.Ca2+ imaging technique was applied to monitor intracellular Ca2+ levels.Patch-clamp technique was used to detect Ca2+-sensitive currents including L-type calcium current(ICa-L)and transient inward current(ITi),and membrane oscillations due to early afterdepolarizations(EADs)or delayed afterdepolarizations(DADs).Western blot was conducted to measure the levels of SR Ca2+ channel ryanodine receptor 2(Ry R2)and its subunits.We compared the changes in intracellular Ca2+ levels,ICa-L,ITi and membrane oscillations under pacing between the R33 Q and the wild type(WT)groups.The effects of Isoproterenol(Iso)and calmodulin dependent protein kinase II(Ca MKII)on Ca2+ triggered activity were also evaluated.Results: In vivo study showed the R33 Q mice were much more susceptible to AF compared with the WT mice(31.57% 6/19 vs 4.56% 1/22,P<0.05)at burst pacing frequency of 25 Hz.Addition of intraperitoneal injection of 1.5mg/kg Iso causeddramatically increases in AF incidences only in the R33 Q group(52.63% 10/19 vs 9.09% 2/22,P<0.01).In vitro study demonstrated the basal intracellular Ca2+ was higher in the R33 Q group compared with the WT group(37.56±4.33 F vs 23.55±1.23 F,P<0.05).There were significant increases in SCa E(11.74% vs 2.24%,P<0.01),ITi(-0.56±0.017 p A/p F vs-0.42±0.014 p A/p F,P<0.05)and membrane oscillations(10.7% vs 4.1%,P<0.05)but there was no significant difference in ICa-L(-3.94±0.53 p A/p F vs-3.42±0.26 p A/p F,P>0.05)in response to pacing frequency of 1 Hz in the R33 Q group compared with the WT group.Addition of 1u M Iso further increased SCa E(from 11.74±0.35% to 17.83±1.72%,P<0.01),ITi(from-0.56±0.017 p A/p F to-0.78±0.03 p A/p F,P<0.05)and membrane oscillations(from 10.7% to 35.7 %,P<0.01)significantly only in the R33 Q group but Iso increased ICa-L in the both groups with more in the R33 Q group(from-3.94±0.53 p A/p F to-9.01± 0.79 p A/p F vs from-3.42±0.26 p A/p F to-7.45±0.37 p A/p F,P<0.05)without functional change of L type calcium channel.Ca MKII inhibition reversed the effects of Iso on RCa E(from 35.7 % to 6.42%),ITi(from-0.78±0.03 p A/p F to-0.61±0.026,P<0.01)and membrane oscillation(from 35.7 % to 14.25%,P<0.01).The increase of SCa E in the R33 Q group was associated with a reduction of SR Ca2+ channel receptor Ry R2 stable subunits including CASQ2(55%),Triadin(63%)and Juctin(30%)rather than the amount of RYR2.Conclusions: This study demonstrates that:(1)the increase of SCa E corresponds to ITi that may trigger membrane oscillations in the R33 Q group,which increase the risk of AF.(2)The occurrence of SCa E in the R33 Q atrial myocytes is due to dysfunction of Ry R2 stable subunits rather than amount of RYR2.(3)Ca MKII inhibitors targeting a reduction of SCa E may be a therapeutic strategy to intervene Ca2+ induced AF associated with R33 Q mutation. |
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