Division of Experimental Cardiology, Medical Faculty Mannheim, University of Heidelberg, Germany.
Circulation. 2012 May 1;125(17):2059-70. doi: 10.1161/CIRCULATIONAHA.111.067306. Epub 2012 Mar 28.
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.
Protein levels (Western blot), membrane currents and action potentials (patch clamp), and Ca(2+) (Fluo-3) were measured in right atrial samples from 76 sinus rhythm (control) and 72 chronic AF (cAF) patients. Diastolic Ca(2+) 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+) 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+) (diastolic Ca(2+)-voltage coupling gain) was higher in cAF compared with control.
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+)-voltage coupling gain, causes AF-promoting atrial delayed afterdepolarizations/triggered activity in cAF patients.
钠钙交换电流（I(NCX)）介导的肌浆网（SR）Ca2+漏反应产生的延迟后去极化（DADs）可促进心房颤动（AF）。导致 AF 患者延迟后去极化的机制尚未明确。
在 76 名窦性节律（对照组）和 72 名慢性 AF（cAF）患者的右心房样本中，测量了蛋白水平（Western blot）、膜电流和动作电位（膜片钳）以及 [Ca2+]（i）（Fluo-3）。cAF 与对照组相比，舒张期 [Ca2+]（i）和 SR Ca2+含量（咖啡因诱导的 Ca2+瞬变期间整合的 I(NCX)）无变化，而通过用四卡因阻断兰尼碱受体（RyR2）估计的舒张期 SR Ca2+漏约增加 50%。在 cAF 样本中，从心房 RyR2 重新构成的脂质双层中的单通道记录显示开放概率增加，为 SR Ca2+漏增加提供了分子基础。钙调蛋白表达（60%）、钙调蛋白依赖性蛋白激酶-II（CaMKII）在 Thr287 处的自身磷酸化（87%）和 RyR2 在 Ser2808 处的磷酸化（蛋白激酶 A/CaMKII 位点，236%）和 Ser2814（CaMKII 位点，77%）在 cAF 中增加。选择性 CaMKII 阻断剂 KN-93 降低了 cAF 中的 SR Ca2+漏、自发性 Ca2+释放事件的频率和 RyR2 开放概率，而 H-89 抑制蛋白激酶 A 则无效。在 RyR2 丝氨酸 2814 处具有组成型磷酸化的基因敲入小鼠中，与对照组相比，Ca2+火花的发生率更高，并且对起搏诱导的 AF 的易感性增加。[Ca2+]（i）和 I(NCX)密度之间的关系表明 cAF 中 I(NCX)上调。与对照组相比，cAF 中更常发生伴有内向 I(NCX)电流的自发性 Ca2+释放事件和延迟后去极化/触发活动，并且静息膜电压对升高的 [Ca2+]（i）（舒张期 [Ca2+]（i）-电压偶联增益）的敏感性更高。
通过 CaMKII-过度磷酸化的 RyR2 增强的 SR Ca2+漏，结合给定的 SR Ca2+释放和增加的舒张期 [Ca2+]（i）-电压偶联增益的更大 I(NCX)，导致 cAF 患者促进 AF 的心房延迟后去极化/触发活动。
